bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhancing the anti‐tumor efficacy of chimeric antigen receptor‐expressing T cells with naturally occurring plant stilbenes
Authors: Soles Andrea , Richardson Elle , Barber Amorette
Published: June 2021

Despite advances in treatment options, cancer remains the second leading cause of death in the United States. Many therapies use immune cells, specifically T cells, for cancer therapy. To enhance tumor recognition by T cells, chimeric antigen receptors consisting of signaling domains fused to receptors that recognize tumor antigens can be created and expressed in T cells. One receptor that is a prospective target for a chimeric antigen receptor is PD1 because the ligands for the PD1 receptor are expressed on many cancer types. Our lab has genetically engineered a chimeric PD1 (chPD1) receptor and has shown that chPD1‐expressing T cells kill tumor cells, secrete inflammatory cytokines, reduce tumor burden, and increase survival in multiple tumor types. A current focus of cancer research is the development of combination therapies that enhance anti‐tumor efficacy without increasing harmful side effects. Recently, a family of naturally occurring plant compounds called stilbenes have been shown to enhance T cell function. Therefore, we tested if stilbenes enhanced chPD1 T cell function in breast cancer. Addition of resveratrol, a stilbene found in berries, grapes, peanuts, and other plants, decreased proliferation, enhanced tumor cell killing, and increased secretion of proinflammatory cytokines (IFNγ, GM‐CSF, TNFα, IL‐17, IL‐2, and IL‐21) from chPD1 T cells. Presence of resveratrol also increased skewing of T cell differentiation to a central memory phenotype through reduction of AKT and mTORc1 signaling, reduced expression of T‐bet and BLIMP‐1 transcription factors, and increased expression of Bcl‐6 and Eomes transcription factors. Hence, combination of resveratrol and chimeric antigen receptor‐expressing T cells may enhance anti‐tumor immune responses through inhibition of the AKT and mTORc1 pathways and skewing to a long‐lived central memory phenotype.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Higher‐Order Clustering of the Transmembrane Anchor of DR5 Drives Signaling
Authors: Fu Tianmin , Pan Liqiang , Zhao linlin , Wu Hao , Chou James
Published: June 2021

Receptor clustering on cell membrane plays important roles in the signaling of many immunoreceptors and this mechanism has previously been attributed to the extracellular and/or the intracellular interactions. Here, we report an unexpected finding that for death receptor 5 (DR5), a Type‐1 receptor in the tumor necrosis factor receptor (TNFR) superfamily, the single‐pass transmembrane helix (TMH) alone in the receptor directly assembles a defined higher‐order structure to drive signaling, and that this higher‐order structure is in turn inhibited by the unliganded extracellular domain. Nuclear magnetic resonance structure of the DR5 TMH in bicelles shows distinct faces of the TMH that mediate trimeric and dimeric interactions, allowing formation of dimer‐trimer interaction networks on membrane. Single TMH mutations that disrupt either trimerization or dimerization, as well as double mutations that disrupt both all abolish ligand‐induced receptor activation. Surprisingly, proteolytic removal of the DR5 extracellular domain, which releases the TMH from physical restraints of the pre‐ligand receptor association, can fully activate downstream signaling in the absence of ligand binding. A dimer‐disruptive, but not trimer‐disruptive mutant, decreases DR5 pre‐ligand association, demonstrating dimerization of DR5 before ligand engagement. Collectively, our data suggest a novel model of receptor signaling in which TMH clustering alone can drive DR5 signal transduction, and binding of ligands or agonistic antibodies at the extracellular domain helps to overcome the pre‐ligand autoinhibition.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Polyphenon E Alters Protein Levels in PC‐3 Cells
Authors: Couming Chris , Carastro L. Michael
Published: June 2021

Prostate cancer (PCa) is the most commonly diagnosed cancer in American men. Polyphenon E (Poly E) is a caffeine‐free standardized green tea extract reported to have chemopreventative activity. However, the mechanisms through which Poly E exhibits chemopreventative activity have not yet been fully elucidated. Our working hypothesis was that Poly E alters expression of specific genes to inhibit PCa cell proliferation. We previously conducted DNA microarray and qRT‐PCR analyses of RNA samples isolated from PCa cells (PC‐3) treated with varying levels of Poly E. The qRT‐PCR analysis assessed CASP‐8 and ATR expression changed not more than 2‐fold under Poly E treatments. The aim of the current study was to determine protein levels by Western blotting. PC‐3 cells were treated with Poly E (0 mg/L, 100mg/L, 200mg/L, and 300mg/L) for 24 hours. Protein was extracted and Western blot analysis was performed for ATR and CASP‐8 proteins. Results from Western Blot analysis for ATR indicated increased protein expression (1.43‐fold) at the 200mg/L Poly‐E treatment level compared to the untreated control, but were essentially unchanged at the 100mg/L and 300mg/L Poly E treatments (0.99‐fold and 0.91‐fold, respectively). These data were consistent with observed qRT‐PCR data. Western blot analysis for CASP‐8 indicated decreased protein expression levels of 0.36‐fold, 0.40‐fold and 0.38‐fold at the 100, 200, and 300 mg/L Poly E treatment levels, respectively. This result exhibited a trend similar to our CASP‐8 qRT‐PCR, but exhibited greater degree of decreases.

Support or Funding Information

This work was supported by a University of Tampa David Delo Research Professor Grant (to L.M.C).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evidence that endurance exercise improves cardiac function in POLG mice
Authors: Hill Blake , Fu Vivian , Ugras George , Prolla Tomas
Published: June 2021

Aging is caused by a variety of factors, but a common thread is impaired function in the organelle central to cellular metabolism and energy production: the mitochondrion. Each cell contains ~500–1000 mitochondria (depending on tissue) and each mitochondrion contains its own DNA (~2–10 copies). The mitochondrial DNA (mtDNA) accumulate mutations with age in several species including humans and it has long been suggested that mitochondria are central to human aging. Highlighting the crucial role of mitochondria to aging, a genetic lesion that increases mtDNA damage causes premature aging. This lesion resulted in a residue substitution (D257A) in the highly conserved exonuclease domain of the sole mammalian mtDNA polymerase, POLG, impairing its proofreading ability. Homozygous knock‐in mice with the POLG‐D257A substitution (POLG mice) show a profound aging syndrome with reduced life span and premature aging including weight loss, decreased subcutaneous fat, alopecia, kyphosis, osteoporosis, anemia, reduced fertility, and cardiac dysfunction. Recently endurance exercise was shown to reverse this aging syndrome. Here the exercise protocol was repeated and followed by measurements of blood and cardiac function. Aging in POLG mice was associated with reduced levels of hemoglobin and white blood cells. Endurance exercise failed to prevent age‐related changes. By contrast, many cardiac parameters as measured by high‐resolution echocardiography showed statistically significant differences between young and old sedentary POLG mice. Statistically significant differences were also found between old POLG mice that were either sedentary or exercised. The results indicate that exercise improves some measures of cardiac dysfunction in POLG mice and suggest that interventions may exist to overcome the deleterious effects of mtDNA damage.

Support or Funding Information

This study was funded by a grant from Breakout Labs to CyteGen Corp.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Influence of SNF1 Gene on Glycolytic Flux Addressed to Aerobic Fermentation (Crabtree Effect) of Saccharomyces cerevisiae
Authors: Carrillo Andres , Dufoo‐Hurtado Miguel David , Madrigal‐Perez Luis Alberto
Published: June 2021

The alcoholic fermentation is a biotechnological process, which has been employed for more than one hundred years. Despite the ample researches carry out about this, there are specific aspects that are still not fully understood, an example of this is the Crabtree effect. The Crabtree effect is a metabolic phenomenon that occurs when the concentration of fermentable sugars in the medium is higher than 0.015% (w/v) and is characterized by the increase of glycolytic flux, an augment in the production of metabolites of fermentation and respiration repression. In spite of the wide quantity of information on the Crabtree effect is still not clear the molecular basis by which originates. It has been proposed that sucrose non‐fermenting protein‐1 (Snf1p) plays a key role in the unleashing of the Crabtree effect. Since in high concentrations of glucose, Snf1p is found dephosphorylated; while in low concentrations of glucose the protein Snf1p is phosphorylated and participate in the de‐repression of genes repressed by glucose. However, it has been suggested that both Snf1p phosphorylated and dephosphorylated have an essential participation in the metabolic transition of fermentative metabolism to the respiratory metabolism. This study aimed to evaluate the influence of the deletion of SNF1 gene in the glycolytic flux addressed to the Crabtree effect of S. cerevisiae. For this purpose, we use the Saccharomyces cerevisiae genetic background BY4742 and its mutant in the gene SNF1 (snf1Δ). Here, we provide evidence that snf1Δ mutants grown in YPD medium supplemented with 10% glucose did not present any effect upon glycolytic flux. In this regard, NADH/NAD+ ratio, extracellular acidification rate and transcription levels of genes HXK2 and PFK1 did not show a significative difference between strains snf1Δ and wild‐type. Surprisingly, the strain with the deletion in the gen SNF1 exhibits an increased oxygen consumption in comparison with wild‐type. The results obtained in this study suggest by first time that even when Snf1p is found dephosphorylated in high concentrations of glucose could be necessary for carrying out the repression of the respiration.

Support or Funding Information

PRODEP partially funded this work (project number: ITESCH‐EXB‐002).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Importance of Hydrophobic Bulk at the Rotor‐Stator Interface of E. coli ATP Synthase
Authors: Dodd Michaela , Pressley Benjamin , Steed Ryan
Published: June 2021

As the primary producer of adenosine triphosphate (ATP), the chemical energy currency of cells, F1Fo ATP synthase is essential for life. Using the H+ electrochemical gradient, the membrane‐embedded Fo complex generates torque on the cytoplasmic F1 complex, which synthesizes ATP from ADP and phosphate. While much is known about the mechanism of ATP synthesis, aspects of Fo functionality, including the roles of interactions between subunit a (stator) and the c10 ring (rotor), remain unresolved. In E. coli, Phe54 and Ile55 on subunit c have been found in a previous study to be important for H+ movement through Fo during ATP‐driven H+ pumping. We show here that cF54C and cI55C mutations cannot pump or passively translocate protons through Fo. ATP‐driven H+ pumping activity was partially restored by systematically adding steric bulk to Cys54 through chemical modifications with methanethiosulfonate derivatives of increasing size; however, similar restoration did not occur for Cys55. Additionally, we used site‐directed mutagenesis to replace Phe54 or Ile55 with hydrophobic amino acids of various size, all of which supported H+ pumping activity. While the chemical modification of Cys54 suggested that hydrophobic bulk at this position is important for H+ pumping, the chemical properties required at these positions remain unclear.

Support or Funding Information

Supported by the North Carolina GlaxoSmithKline Foundation and the University of North Carolina Asheville Undergraduate Research Program

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of the SNF1 Deletion in the Glycolytic Pathway of Saccharomyces cerevisiae Grown at 1% Glucose
Authors: Martinez‐Ortiz Cecilia , Dufoo‐Hurtado Miguel David , Madrigal‐Perez Luis Alberto
Published: June 2021

The alcoholic fermentation is an amphibolic process that allows the formation of ATP at substrate‐level, keeps active the glycolysis through re‐oxidation of NADH, and generate metabolites of biotechnological interest. Saccharomyces cerevisiae is a positive Crabtree, which performs the alcoholic fermentation in oxygen presence, increasing the glycolytic flux accompanied by repression of respiration. However, the molecular mechanism of the Crabtree effect is still not clear. The sucrose non‐fermenting protein‐1 (Snf1p) is a cellular energy sensor that modulates the switch between respiration and alcoholic fermentation. Therefore, Snf1p could be a key piece in the molecular puzzle of the alcoholic fermentation related to the Crabtree effect. For this reason, this work aimed to determine whether the deletion in SNF1 gene affects the alcoholic fermentation at 1% glucose. For this purpose, the BY4742 genetic background was used and its mutant in the SNF1 gene. In this way, it was found: 1) that the absence of SNF1 gene affects growth. 2) A decrease in extracellular acidification rate in SNF1 mutant. 3) NADH/NAD ratio does not have any change in the strains. However, the amount NAD(P)H showed an increase in SNF1 mutant. 4) Transcription levels of HXK2 gene displayed an increase in SNF1 mutant; while transcription levels of PFK1 gene decrease. 5) The basal respiration and the maximum respiratory capacity diminished in SNF1 mutant. All data suggest that deletion in SNF1 gene favors the formation of the anabolic species NADPH, probably by a transcriptional regulation of glycolysis, not observing an effect in the alcoholic fermentation.

Support or Funding Information

PRODEP partially funded this work (project number: ITESCH‐EXB‐002).

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 A2B Adenosine Receptor Antagonist Alloxazine Exacerbates NASH and Disrupts Cellular Folate Metabolism in Methionine and Choline Deficient Mice
Authors: Eudy Brandon James , Silva Robin da
Published: June 2021

Nonalcoholic fatty liver disease is characterized by hepatic lipid accumulation of greater than 5% and is a rising healthcare problem in the United States. Approximately 25–30% of the US population may have a fatty liver and 10–15% of these patients will progress to more severe disease known as nonalcoholic steatohepatitis (NASH). There are no approved treatments for NASH and the factors that result in the transition from fatty liver to NASH are not well defined. Folate and one‐carbon metabolism are implicated in the pathogenesis of NASH and are important pathways for the biosynthesis of many important molecules like creatine, phosphatidylcholine, purines, and thymidine. Additionally, the purine adenosine modulates immune cells by signaling to cell surface receptors on macrophages in the liver. In the present study, mice were fed a methionine and choline deficient diet to induce NASH lesions and treated with a putative A2B adenosine receptor antagonist, alloxazine, to probe the role of this adenosine receptor in NASH. MCD diet feeding reduced purine and thymidine nucleotides in the liver; potentially limiting the availability of crucial molecules for regeneration of liver tissue. MCD mice showed reduced activating transcription factor 4 (ATF4) expression, which has been shown to regulate mitochondrial folate metabolism and formate production which is required for purine biosynthesis. Alloxazine treatment exacerbated hepatic lipid accumulation and caused severe lobular inflammation in MCD mice. Alloxazine also disrupted hepatic folate metabolism and reduced the concentration of formate without altering concentrations of S‐adenosylmethionine and S‐adenosylhomocysteine. These data suggest that in addition to its function as an A2B adenosine receptor antagonist, alloxazine also impairs cellular folate metabolism. These data provide a novel link between folate and lipid metabolism, and inflammation in NASH.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterizing the Lone Acyltransferase Homolog in Saccharomyces cerevisiae
Authors: Ullrich Alexander , Hardin Blake , Khanafer Adam , Oelkers Peter
Published: June 2021

Cell membranes are largely composed of glycerophospholipids (PL) that create a barrier and separate intracellular aqueous environments. PL are heterogeneous, amphipathic lipids containing a glycerol backbone. In Saccharomyces cerevisiae, our previous work found that 108 different PL structural species were maintained at consistent relative abundance. Our aim is to understand the molecular mechanisms that maintain and regulate PL species abundance. The distribution of acyl chains in PL may be controlled by the activity and substrate specificity of acyltransferases which use an acyl‐CoA substrate to attach a hydrocarbon chain to an acceptor's hydroxyl group via an ester bond. In S. cerevisiae, at least 11 characterized acyltransferases contribute to PL metabolism. One, PSI1p, which esterifies 1‐hydroxy‐2‐acyl phosphatidylinositol with stearoyl‐CoA, has an uncharacterized paralog, YDR018c. Determining the acyl acceptor for YDR018c will likely clarify mechanisms for PL homeostasis in S. cerevisiae and provide a functional precedent for its most similar human homologs, 1‐acyl‐3‐glycerophosphate acyltransferase (AGPAT) 3 and 4. Since Psi1p has been shown to utilize an acyl acceptor with a large head group, inositol, it is possible that YDR018c utilizes a similar acyl acceptor.

Since work by others and our lab did not identify a PL phenotype for haploid S. cerevisiae harboring a targeted deletion of YDR018c, loss of its function may be masked by the activity of other acyltransferases. To address this, we have made compound, targeted gene deletions of YDR018c, PSI1 and combinations of glycerol‐3‐phosphate O‐acyltransferases (GPATs; GAT1 or GAT2) and AGPATs (SLC1 or LPT1). Growth phenotype assays, including temperature sensitivity and providing various metabolic stresses, are ongoing to identify physiological changes in the compound deletion strains. Phospholipidome analysis, after culturing the strains in conditions shown to elicit a growth phenotype, will follow so to identify the absence or loss of specific PL species in conjunction with YDR018c deletion.

Support or Funding Information

This project was supported by the 2018 Summer Undergraduate Research Experience (SURE) Program from the University of Michigan‐Dearborn's Office of Research and Sponsored Programs (ORSP).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of genes responsible for cholesterol uptake and efflux by fatty acids in THP‐1 macrophages
Authors: Moctar Khadija , Rasiah Angela , Medh Jheem D.
Published: June 2021

Cholesterol is a very hydrophobic molecule that is transported through the blood in complexes containing apolipoproteins. Lipoprotein lipase (LPL) is a secreted enzyme that localizes to the luminal side of blood vessels. LPL is proatherogenic and facilitates occlusion of arterial tissue. When there is an abundance of lipids in the body, cytokines stimulate the release of vascular cell adhesion molecules (VCAM) by endothelial cells lining the arteries. This response induces the adhesion of monocytes to endothelial cells allowing monocytes to pass through gaps in the endothelium into the bloodstream. The monocytes take up oxidized lipids and differentiate to macrophages. If these macrophages are unsuccessful at eradicating lipid particles, they become foam cells and build up as plaques lining arteries.

There are certain receptors and transporters that take part in cholesterol homeostasis. Scavenger receptors (CD36, SR‐AI, SR‐BI) mediate the cellular uptake of cholesterol whereas the ATP‐binding cassette transporter A1 (ABCA1) mediates the unidirectional efflux of excess cholesterol. SR‐BI also mediates bidirectional cholesterol transport.

It has been reported in the literature that unsaturated fatty acids inhibit ABCA1 expression. This led us to ask, do the products of normal LPL function affect ABCA1 gene expression levels? To investigate this, we differentiate THP‐1 macrophages and stimulated them with various saturated and unsaturated fatty acids, namely, oleic acid a monounsaturated omega‐9 fatty acid, linoleic acid a polyunsaturated omega‐6 essential fatty acid and stearic acid, a common saturated fatty acid. The expression levels of CD36/SRA1 and SRB1 protein levels were compared in treated and untreated cells by Western blot analysis.

Support or Funding Information

This work was supported by National Institutes of Health Awards R15HL083946, SC3GM095413, and GM063787.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lipidomic and proteomic evaluation of extracellular membrane vesicles from Streptococcus mutans wild‐type, ΔsrtA and Δsfp strains
Authors: Aparicio Joyce Corina Morales , Berdon Ana Lilia Barran , Green Kari B. , Wen Zezhang , Brady L. Jeannine
Published: June 2021

Background

Extracellular membrane vesicles (EMVs) have long been studied in Gram‐negative bacteria. It is more recently established that Gram‐positive bacteria are capable of producing EMVs despite their thick cell wall. Our group showed that the cariogenic bacterium Streptococcus mutans produces EMVs that carry protein and eDNA. EMV protein content is altered in a deletion mutant lacking the transpeptidase, Sortase A. In Bacillus subtilis, EMVs are disrupted by the lipoprotein surfactin and accumulate in a Δsfp mutant. Our current objective is to isolate EMVs from wild‐type, ΔsrtA and Δsfp strains in order to compare their respective lipidomic and proteomic profiles, as well as to compare each strain's EMVs with corresponding cytoplasmic membrane preparations.

Methods

EMVs were isolated from four separate replicates of each strain grown in Terleckyj's defined media (TDM). Filtered culture supernatants were concentrated ~50 fold, and EMVs were harvested by high‐speed centrifugation and density gradient isolation. Cytoplasmic membranes were isolated from protoplasts derived from bacterial cells from the same cultures. Lipids were analyzed by capillary‐liquid chromatography mass spectrometry (Cap‐LC‐MS). Proteins were analyzed by Nano‐Liquid chromatography tandem mass spectrometry (Nano‐LC/MS/MS). MV size, concentration, and polydisperity were assessed by transmission electron microscopy, nanoparticle tracking analysis, and dynamic light scattering.

Results

EMVs from the ΔsrtA mutant were enlarged compared to the other strains. Lipid composition of EMV membranes consisted primarily of glycerophospholipids, with some glycerolipids and sphingolipids. Glycerophosphoinsitol (PI) was underrepresented in both mutant strains compared to the WT. Glycerophosphocholine (PC) was overrepresented in the Δsfp mutant compared to the other two strains. Cardiolipin (CL) was overrepresented in EMVs from the WT strain compared to its corresponding cytoplasmic membrane preparations. Proteomics analyses are underway.

Conclusions

Lipidomic and proteomic analyses will provide information regarding S. mutans vesiculogenesis and potential functional differences in EMVs produced by WT and mutant strains.

Support or Funding Information

NIH R21DE025348 NIH R01DE008007

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mutations in the Human Follicle Stimulating Hormone Receptor Caveolin Binding Motif Cause Increased Basal Activation
Authors: Altman Elizabeth K , Cohen Brian D
Published: June 2021

Over twelve percent of women aged fifteen to forty‐five in America suffer from infertility and over seven million women have used infertility services, such as intrauterine insemination and in vitro fertilization. Some cases of infertility may be due to dysfunctional human follicle stimulating hormone (hFSH) signaling. hFSH plays a role in spermatogenesis in males, as well as follicle maturation and estrogen production in females. Problems with either hFSH or the hFSH receptor (hFSHR) decrease fertility in males and cause complete infertility in females. As part of the hypothalamic‐pituitary‐gonadal axis, hFSH is released from the pituitary gland and binds to hFSHR in the ovaries and testes. It has been shown that hFSHR interacts with lipid rafts, stiffer regions of the plasma membrane with higher sphingolipid and cholesterol concentrations, and with caveolin, a protein found in certain lipid rafts. This interaction may occur through a specific sequence of amino acids in the 4th transmembrane domain of the hFSHR consistent with a caveolin binding motif (CBM). Previous work from our lab also suggested that hFSHR signaling is regulated through residency in lipid rafts, such that hFSHR signaling is inhibited as a result of recruitment into lipid rafts. The current study investigated the effect of mutations of the CBM on hFSHR signaling. It was hypothesized that complex mutation of the CBM would cause increased hFSHR signaling by disrupting interactions with caveolin, preventing inhibition via residency in lipid rafts. Mutants were created using site‐directed mutagenesis and expression vectors were transiently transfected into HEK293 cells and hFSHR signaling was qualitatively compared between mutants via western blot. Mutation of two sites in the CBM resulted in increased basal and induced hFSHR signaling, yet mutation of three sites decreased signaling below that of the wild type receptor. Mutation of two sites supports the hypothesis, while the decreased signaling from the triple mutant may suggest too large of a conformational change to allow for any receptor function. Further understanding of hFSHR signaling regulation would allow for development of new fertility treatments for men and women.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Direct Imaging of the Cholesterol and Sphingolipid Abundance at the Site of Influenza Virus Assembly with High‐Resolution SIMS
Authors: Kraft Mary L , Yeager Ashley N , Weber Peter K , Joshua Joshua
Published: June 2021

The influenza virus is hypothesized to assemble and bud from plasma membrane domains that are enriched with cholesterol and sphingolipids. This hypothesis is consistent with the reported enrichment of the influenza viral envelope with cholesterol and sphingolipids, the reduction in influenza virus protein clustering in the plasma membrane induced by cholesterol depletion, and the sensitivity of influenza virus formation to cellular cholesterol levels. Nonetheless, the levels of cholesterol and sphingolipids in the plasma membrane at the site of influenza virus budding have never been directly measured. The main challenge has been a lack of analytical methods with the sensitivity and lateral resolution that is required to measure the relative cholesterol and sphingolipid levels in the plasma membrane on the 100‐nm‐length scales that are relevant to influenza virus budding. We recently overcame this challenge by using a secondary ion mass spectrometry (SIMS) technique with high spatial resolution and sensitivity to image the distributions of stable isotope‐labeled cholesterol, sphingolipids, and immunolabeled influenza hemagglutinin in the plasma membranes of virus‐infected MDCK cells. Our results show that the plasma membranes of both influenza‐infected and uninfected MDCK cells contained regions that were enriched with sphingolipids, but cholesterol appeared to be uniformly distributed within the membrane. Examination of the influenza‐infected MDCK cells revealed the hemagglutinin‐rich sites of influenza virus assembly in the plasma membrane were not enriched with cholesterol, and seldom enriched with sphingolipids. These direct imaging results demonstrate that the influenza virus does not assemble within plasma membrane domains that are enriched with cholesterol and sphingolipids.

Support or Funding Information

This research was supported by the U.S. National Science Foundation under CHE 15‐08662.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural and biochemical analysis completes the puzzle of chitin hydrolysis in insects
Authors: Yang Qing , Chen Wei
Published: June 2021

Chitin is a linear homopolymer of β‐N‐acetyl‐D‐glucosamines and a major structural component of insect cuticles. Chitin hydrolysis involves glycoside hydrolase family 18 (GH18) chitinases and GH20 β‐N‐acetyl‐D‐hexosaminidases. In insects, chitin hydrolysis is essential for periodically shedding of the old cuticle ecdysis and proceeds via a pathway different from that in the well‐studied bacterial chitinolytic system. In Lepidopteran insects, the group II chitinase (ChtII) together with two other chitinases (ChtI and Chi‐h) and one GH20 β‐N‐acetyl‐D‐hexosaminidase (Hex1) are essential (Fig.1). Having revealed the acting mode and catalysis of ChtI, Chi‐h and Hex1, our lab recently elucidated the role of ChtII. ChtII is a widespread chitinolytic enzyme in insects and contains the greatest number of catalytic domains and chitin binding domains. We investigated the structure and enzymology of OfChtII, a ChtII representative derived from the insect pest Ostrinia furnacalis. OfChtII exhibit structural characteristics within the substrate‐binding cleft similar to those in OfChi‐h and OfChtI. However, it lacks structural elements favoring substrate binding beyond the active sites, including an extra wall structure present in OfChi‐h. Nevertheless, the numerous domains in OfChtII may compensate for this difference; a truncation containing one catalytic domain and three chitin binding modules (OfChtII‐B4C1) displayed higher activity toward insoluble polymeric substrates than those of OfChi‐h and OfChtI. Our observations complete the puzzle of chitin hydrolysis in insects and suggest a unique system distinguished from the bacterial ones. And based on the structural uniqueness, we have been able to develop highly efficient inhibitors against OfChi‐h, OfChtI, OfChtII and OfHex1.

Support or Funding Information

This work was supported by the Program for National Natural Science Funds for Distinguished Young Scholar (31425021), the National Natural Science Foundation of China (31402015), the National Key Research and Development Project of China (2017YFD0200502, 2017YFD0201207), the Fundamental Research Funds for the Central Universities (DUT17RC(4)13) and China Postdoctoral Science Foundation funded project (2017M611234).

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 New Buzz about Sugars: Novel N‐Glycans in Bees, Moths and Mosquitoes
Authors: Wilson Iain B. H. , Hykollari Alba , Eckmair Barbara , Malzl Daniel , Vanbeselaere Jorick , Paschinger Katharina
Published: June 2021

Sugars cover the surfaces of all cells and a major class of these glycoconjugates are the asparagine‐linked oligosaccharides attached to glycoproteins (N‐glycans). The classical view has been that N‐glycans in ‘lower’ are simple. However, recent data shows that, other than plants, the diversity of N‐glycan modifications in non‐vertebrate species is extremely high. Based on isobaric/isomeric separation combined with chemical or enzymatic treatments and MALDI‐TOF MS/MS, our own studies on dipteran, lepidopteran and hymenopteran species (specifically mosquitoes, moths and the honeybee) reveal a variety of ‘charged’ modifications of N‐glycans from insects, such as glucuronic acid, sulphate, phosphoethanolamine and phosphorylcholine. In addition, unusual core mannosylation, variable antennal length as well as up to three branches increase the range of possibilities. In the honeybee, we also have evidence for tissue‐specific N‐glycosylation patterns when comparing larvae, royal jelly and venom, while immunomodulatory or immunogenic epitopes are found on N‐glycans from insect cell lines used as baculovirus expression hosts. It is also clear that each analysed insect species has its own N‐glycosylation profile due to variations in relative abundance or absence of certain glycan structures. Complementary to the MS analyses, binding to antibodies, lectins and pentraxins can be tested with either natural N‐glycan pools in an array format or glycoproteins on blots; thereby, interactions with proteins of the human innate immune system can be demonstrated. Our data not only show that the concept of ‘simple’ organisms possessing only ‘simple’ glycans is not a true reflection of the actually complex biosynthetic glycosylation capacity of insects, but have repercussions for the use of insect cell lines for recombinant protein expression.

Support or Funding Information

This work was supported by the Austrian Science Fund (FWF) by grants to K.P., A.H. and I.B.H.W.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of O‐GlcNAc Hydrolase with Phosphomimetic Mutations in HeLa Cells
Authors: Hinkle Megan Leigh , Crawford Garland
Published: June 2021

O‐linked beta N‐acetylglucosamine (O‐GlcNAc) hydrolase, or OGA, is an enzyme involved in a post‐translational modification. This enzyme hydrolyzes a single O‐GlcNAc from serine or threonine residues on target proteins located in the cytosol of metazoan organisms. OGA is the only protein known to carry out this function in the context of cytosolic proteins. This exclusivity of function makes OGA a likely target of post‐translational modifications in order to regulate its activity. Our aim is to generate a phosphomimetic OGA using site‐directed mutagenesis to convert a single serine or threonine to an aspartic acid and to introduce the mutated OGA gene into HeLa cells to study the relationship between OGA phosphorylation and its activity. While multiple serine and threonine residues of OGA have been predicted by bioinformatics and experimental approaches to be phosphorylated, the phosphorylation of OGA at serine 364 is of particular interest. This residue has been identified to be phosphorylated in 45 separate experiments and the introduction of OGA with a mutation from a serine to an aspartic acid residue at this point shuts down bacterial growth. Our aim is to study the effect of the phosphomimetic mutation at serine 364 in a mammalian cell system. Analysis by Western blot will reveal the extent to which the mutants alter OGA expression levels and O‐GlcNAc modification levels in the cell. Additionally, interactions of OGA with known binding partners, such as OGT, the enzyme which adds the O‐GlcNAc sugar to target proteins, can be studied with co‐immunoprecipitation. This work lays the foundation for identifying potential phosphorylation states of OGA and would aid in elucidating the regulatory pathways of this enzyme.

Support or Funding Information

SEED grant, Office of the Provost, Mercer University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cellulases Specific Activities Study of Enzymes secreted by Fusarium sp. and Phlebia sp. in Aerobic and Anaerobic Conditions
Authors: Mercado Benjamin
Published: June 2021

Cellulose is the most abundant organic compound on Earth but we can't use it plenty as raw material due to its molecular arrangement and composition. Cellulases are powerful enzymes secreted by some organisms that can be used to degrade cellulose into simple organic molecules like glucose or cellobiose. Although traditional filamentous fungi are known to be excellent cellulose degraders in aerobic conditions, there is little known about cellulose degradation by fungi in anaerobic condition. In this study two facultative anaerobic filamentous fungi isolated and identified using ITS were used to compare cellulase specific activities in aerobic and anaerobic conditions. One Fusarium sp. and Phlebia sp. were grown individually in Potato Dextrose agar for 7 days. Suspensions of spores were prepared for each organism and use it as inoculums. Liquid media culture was prepared and enriched with Carboxylmethylcellulose, Wheat Brand Flour and Whatman no.1 filer paper and inoculated with 1mL of 106 suspension spores of each fungi individually. Cellulase specific activities were determined using Dinitrosalicylic acid method at ph 4, 5, 6, and 7 at 30°C, 40°C, 50°C and 60°C after one month of incubation. Fusarium sp. showed a maximum of cellulase activity at 40°C and pH 5 for both aerobic and anaerobic conditions with values of 0.436 and 0.403 respectively. On the other hand Phlebia sp. showed better results in anaerobic condition with a value of 0.715 at 60°C and pH 6 against a value of 0.632 in aerobic condition at 60°C and ph 5. These results confirm different trending in cellulase specific activities in aerobic and anaerobic conditions and could suggest the presence of different enzymes secreted depending of oxygen concentrations.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Unraveling the activity of glycosyltransferases and other PTM enzymes with bioluminescent biochemical and cell based assays
Authors: Zegzouti Hicham , Engel Laurie , Hwang Brian (Byounghoon) , Alves Juliano , Goueli Said
Published: June 2021

Post translational modifications (PTMs) are central to all aspects of biological regulation. PTMs amplify the diverse functions of the proteome by covalently adding functional groups to proteins. These modifications include phosphorylation, methylation, glycosylation, and ubiquitination, and influence many aspects of normal cell biology and pathogenesis. Examples of post translational modification enzymes include but are not limited to, kinases/phosphatases, methyltransferases/demethylases, and glycosyltransferases/glucanases. Under normal physiological conditions, the regulation of PTM enzymes is tightly regulated. However, under pathological conditions, these enzyme activities can be dysregulated, and the disruption of the intracellular networks they govern leads to an array of diseases including cancer and inflammation. Consequently, PTM enzymes have become important targets for drug discovery creating a need for development of activity detection assays. Current assays to analyze cellular or biochemical PTM enzyme activities can be tedious, non‐homogeneous, and not easily adaptable to HTS. They can also suffer from generation of false hits due to compound interference or require special instruments for detection. To overcome these shortcomings, we developed an array of biochemical and cellular bioluminescent assays to detect these enzyme activities. This presentation will showcase how cellular mechanisms driven by PTM in general and phosphorylation and glcnacylation in particular can be addressed by analyzing these enzyme's cellular activity using the NanoLuc Binary Technology (NanoBiT), a two‐subunit complementation system based on NanoLuc luciferase. We will also discuss how the bioluminescent nucleotide assays that detect UDP, GDP, UMP and CMP as means to measure glycosyltransferase activities can streamline studies on GT specificity of transfer of different sugars to different acceptors. We will show their use in screening for specific inhibitors as well as in studies of their modes of action. Development of these bioluminescent detection assays will enable the investigation of key signaling pathways and the study of many PTM enzymes in general and GTs in particular, which may have significant impact on diverse areas of glycobiology research.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
When Wolff's law isn't enough: training students to consider soft tissues in promoting skeletal health
Authors: Hamrick Mark
Published: June 2021

The physiology of various organ systems has revealed extensive crosstalk among tissues such as adipose tissue, skeletal muscle, and bone. These findings have important implications for skeletal biology, particularly within the context of improving bone health during growth and with aging. The latter is especially significant for students in the health sciences, since the population over the age of 65 is the most rapidly growing demographic globally and in the United States. Mechanical loads emanating from body weight and muscle contraction are important for bone mass accrual early in life, but the response of bone to mechanical stimuli is markedly attenuated in adults. Though increased loading magnitude and frequency may have less dramatic anabolic effects on the adult skeleton than on young bones, inactivity and muscle disuse atrophy can contribute significantly to age‐related bone loss. This point is reinforced by data from clinical studies showing that older adults with low appendicular lean mass and reduced grip strength also have reduced cortical bone area and thickness. The highly integrated changes in skeletal muscle and bone observed in aging patient populations suggest that targeting either sarcopenia or osteoporosis alone may not be sufficient for effective fracture prevention. Thus, new strategies for improving both tissues simultaneously to prevent “osteosarcopenia” have been proposed. These recent advances underscore the importance of considering soft‐tissues for maintaining bone health and preventing falls and bone fractures.

Support or Funding Information

National Institute on Aging (AG 036675)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Rationally Engineered Hyperactive Actin‐Resistant DNase1‐Fc Fusion Protein Ameliorates Autoimmune Glomerulonephritis
Authors: Austin Cary D. , Mouchess Maria , Suto Eric , Chan Sara , Dai Bingbing , Santell Lydia , Tam Christine , Hackney Jason A. , Cao Yi , Luan Peng , Darwish Martine , He Dongping , Haller Susan , Eastham‐Anderson Jefferey , Franke Yvonne , Lu Yanmei , Blanchette Craig , Lee Wyne P. , Lazarus Robert A. , Yi Tangsheng
Published: June 2021

Loss of B cell tolerance and generation of autoreactive anti‐nuclear antibodies are hallmarks of systemic lupus erythematosus (SLE) and lupus nephritis. Lupus nephritis is characterized by glomerular and tubulointerstitial inflammation often initiated by the renal glomerular deposition of anti‐nuclear immune complexes which trigger subsequent activation of complement, macrophages/monocytes and other innate inflammatory cells. The mechanism of anti‐nuclear immunoglobulin accumulation and clearance in lupus nephritis pathogenesis remains largely uncharacterized. Here, we show that innate immune activation in the NZB/W F1 mouse model and in human lupus nephritis biopsies rapidly reduces DNase1 expression in renal cortex proximal tubular cells. To overcome the loss of endogenous DNase1, we treated lupus‐prone mice with a hyperactive actin resistant variant of DNase1 with improved catalytic activity against nucleic acid‐IgG immune complexes and acceptable in vivo pharmacokinetics. Hyperactive DNase1‐Fc fusion protein ameliorates nephritis in a murine model of lupus nephritis and reduces immune complex deposition/complement fixation. Taken together, our data suggest that the loss of renal DNase1 through TLR signaling or other innate immune activation impairs clearance of autoreactive anti‐nuclear immune complex deposits in the kidney to promote nephritis progression. Our findings provide a therapeutic rationale for using an engineered DNase1‐Fc as a potential therapeutic approach in lupus nephritis.

Support or Funding Information

All authors are current or previous employees of Genentech, Inc.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of gene augmentation strategy for the treatment of MAK associated Retinitis Pigmentosa
Authors: Gibson‐Corley Katherine N. , Cranston Cathryn , Anfinson Kristin , Kaalberg Emily , Han Ian , Slusarski Diane C. , Goeken Adam , Businga Thomas , Brown Hannah , Mullins Robert F. , Stone Edwin M. , Tucker Budd A.
Published: June 2021

Purpose

The gene male germ cell associated kinase (MAK) encodes a protein involved in regulation of photoreceptor cilia length. By combining next generation whole exome sequencing and induced pluripotent stem cell (iPSC) technology we found that an Alu repeat inserted in exon 9 of the MAK gene resulted in a loss of normal MAK transcript and development of human autosomal recessive retinitis pigmentosa (RP). Although a relatively rare cause of disease in the general population, the MAK variant is enriched in individuals of Ashkenazi Jewish descent. In this population, 1 in 55 individuals are carriers and 1/3 of all cases of recessive RP is caused by this gene. The purpose of this study was to determine if a viral gene augmentation strategy could be used to restore functional MAK protein and in turn would be a good candidate for treatment of early stage MAK associated RP.

Methods

To demonstrate the ability of the developed MAK gene transfer vector to restore normal transcript and protein, patient specific iPSC derived photoreceptor precursor cells were transduced and immunocytochemistry, rt‐PCR and western blot analysis were performed. To demonstrate transgene function, cilia length assays were performed using patient derived fibroblast cells in vitro and MAK knock out zebra fish in vivo. In addition, visual function testing was performed in the zebra fish. To demonstrate safety of cGMP generated clinical grade vector, local and systemic toxicity studies were performed via subretinal injection into wildtype rats.

Results

MAK mutant RP iPSC derived photoreceptor cells harboring the previously identified ALU insertion (Tucker et. al. 2011) were generated and transduced with viral vectors containing the retinal MAK transcript. At 1‐week post‐transduction normal retinal MAK transcript and protein could be detected via rt‐PCR and western blotting respectively. Using patient derived fibroblast cells and MAK knockdown zebra fish we demonstrate that over expression of retinal MAK transgene restored primary cilia length. In addition, the visual defect identified in MAK knockdown zebra fish was mitigated via treatment with retinal MAK transgene. Following subretinal delivery of clinical grade vector into wild type rats no evidence of local or systemic toxicity was detected at either 1 or 3 months' post‐treatment.

Conclusions

We have successfully developed a MAK gene replacement strategy that has been validated in human iPSC derived photoreceptor precursor cells in vitro and MAK knock down zebra fish and wildtype rats in vivo.

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