bluuebunny/crossref_metadata_2025 huggingface dataset:
Resveratrol and CXCR4 Mediated Migration of Brest Cancer Cell Lines
Authors: Cruz Kevin , Padilla Luis , Trossi Geraline , Muñoz Kevin , Robles Jose , Arroyo Gerardo , Suarez Edu , Ruiz Abigail
Published: June 2021

In the United States, breast cancer is one of the most common types of cancer among women, being the second cause of cancer‐related deaths. Despite early detection methods and available treatments, breast cancer incidence is increasing worldwide. Some malignant cells have the potential to migrate and invade, metastasizing to distant organs and tissues. Current treatments include surgery, chemotherapy, and radiotherapy. These treatments usually produce side effects that have a negative influence in the quality of life of the patients. Additional studies are necessary to identify candidate natural compounds targeting specific molecules and receptors expressed in breast cancer cells. For example, CXCR4 is a G protein‐coupled receptor known to be one of the major proteins involved in metastasis of breast cancer. It mediates migration towards tissues expressing its specific ligand CXCL12. Natural compounds such as resveratrol, a plant polyphenol commonly found in grapes, peanuts and berries, among other, has shown anti‐proliferative, anti‐estrogenic, and antioxidant properties. We aimed to study the effect of resveratrol on CXCR4‐mediated cell migration. We hypothesize resveratrol will inhibit the ligand‐receptor relation between CXCR4 and CXCL12 decreasing the migration. To accomplish this, we conducted a wound healing assays, in Hs578t breast carcinoma‐cell line and MCF 7 breast adenocarcinoma‐cell lines treated with 25, 50, 100, and 200 μM of resveratrol in the presence of 100 ng/mL of CXCL12. As an internal control we used AMD3100, an antagonist of CXCR4. The results on Hs578t carcinoma cells did not show significant differences in the migration compared to the controls. However, this preliminary data suggest that there is a decrease in migration in the cells treated with 25 and 100 μM of resveratrol, compared to the AMD3100. Regarding MCF‐7 adenocarcinoma cells, we did not observe significant differences in cell migration. Additional experimental replicates are necessary to validate these results. In addition, we are currently studying other malignant and non‐malignant cell lines, including: CCD‐1097 and CCD‐1074 to identify cell specific responses to resveratrol, mediated by the CXCR4‐CXCL12 signaling pathway.

Support or Funding Information

NIH‐NIGMS #2R25GM096955

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Non‐Canonical Wnt/Calcium Pathway Regulates the Migration and Function of Primary Mesenchyme Cells
Authors: George Alexander , McCann Tyler , Song Jia
Published: June 2021

The highly conserved non‐canonical Wnt/Calcium Signaling Pathway (ncWnt/Ca2+) has been shown to regulate cell motility and epithelial to mesenchymal transition, implicating its role in various developmental processes and cancer invasiveness. We use the sea urchin Primary Mesenchyme Cells (PMCs), the only skeletogenic cells in developing sea urchin embryos, to examine directed cell migration. The working hypothesis is that PMC migration and PMC skeletal function are in part regulated by the ncWnt/Ca2+ signaling pathway. We have shown that disruption of elements downstream of the ncWnt/Ca2+ pathway using perturbative drugs decreased embryonic skeletal length as well as dramatically altered PMC migration patterns. In addition to producing similar skeletal and migratory defects, activation of Protein Kinase C (PKC) downstream of the ncWnt/Ca2+ pathway by Phorbol 12‐myristate 13‐acetate (PMA) also resulted in altered spatial expression of transcripts known to regulate PMC migration and function, as well as changed the expression levels of biomineralization genes. Similar phenotypes were observed in PMA treated embryos and the microRNA‐1 (miR‐1) knockdown embryos. MicroRNAs (miRNAs) are short, non‐coding RNAs that suppress gene expression by binding to the 3′UTR of target mRNA transcripts to repress translation. MiR‐1 is bioinformatically identified to bind to PKC and cdc42 of the ncWnt/Ca2+ signaling cascade, indicating its potential role in regulating the ncWnt/Ca2+ signaling pathway. This study identifies the molecular mechanisms of how the ncWnt/Ca2+ pathway impacts PMC directed migration and developmental function which may potentially contribute to our understanding of birth defects and cancer metastasis.

Support or Funding Information

‐Senior Thesis Winter Session Scholars Award (Fall 2017)

‐Undergraduate Research Supplies‐And‐Expense Grant (Fall 2016, Fall 2017)

‐Science and Engineering Summer Scholar Award (Summer 2017)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Crosstalk Between Tetraspanin‐Interacting Protein Igsf3 and Sphingolipid Metabolism
Authors: Schweitzer Kelly , Ni Kevin , Petrache Irina
Published: June 2021

Rationale

Building on our finding that the tetraspanin‐interacting protein Igsf3 controls glucosylceramide levels in lung endothelial and epithelial cells, we investigated the mechanism and functional impact of Igsf3 regulation of sphingolipid metabolism. We focused on tetraspanin‐controlled cell functions, such as cell migration and adhesion and effectors such as the tight junction proteins, including claudins.

Methods

Primary rat lung endothelial cells and human bronchial epithelial cells (Beas2b) were stably transfected with lentiviral IGSF3 shRNA (R‐shIGSF3 and B2B‐shIGSF3, respectively) or control shRNA or with pIGSF3‐V5His overexpression plasmid. Primary human lung microvascular cells (HMVECL) were treated with IGSF3 siRNA or non‐targeting siRNA. Gene expression profiles were investigated using TaqMan Low Density Arrays (TLDA) for genes involved in the sphingolipid metabolism in epithelial cells with IGSF3 knockdown and in lymphocytes lacking IGSF3. Cell migration and cell adhesion assays were performed using standard methods.

Results

Loss of IGSF3 in B2B‐shIGSF3 and in lymphocytes, in parallel with a dysregulation of glucosylceramide levels, was associated with significant increases in Dihydroceramide Desaturase 1 (DEGS1, p<0.05, p<0.0001), Neutral Sphingomyelinase (SMPD2, p=0.07, p<0.05), Acid Ceramidase (ASAH1, p<0.0001, p<0.01), and Glucosylceramide Synthase (UGCG, p<0.01; ns) mRNA. Functionally, all approaches to decrease IGSF3 caused a significant delay in cell migration, whereas cells overexpressing IGSF3 had increased migration compared to controls (p<0.001). The decrease in migration was attenuated with pharmacological inhibition of Ugcg (p<0.001). In addition, adherent or non‐adherent cells with decreased IGSF3 expression exhibited enhanced cell adhesion to fibronectin (p<0.0001) or to other cells (p<0.01), respectively, which was attenuated with pharmacological inhibition of Asah1 (p<0.05) and was associated with increased expression of claudin 6 (CLDN6, p<0.05, p<0.05, respectively), and decreased expression of claudin 7 (CLDN7, p<0.001, p<0.0001, respectively) mRNA.

Conclusions

The tetraspanin‐interacting protein IGSF3 promotes cell migration and inhibits cell adhesion through inhibition of glucosylceramide synthase and acid ceramidase, respectively. Conditions characterized by loss of IGSF3, through an upregulation of sphingolipid production towards glycosphingolipids and decreased cell migration, may impair functions that require proper cell migration, such as lung tissue repair.

Support or Funding Information

Funding: Wollowick Chair of Pulmonary Research, Department of Medicine Research Microgrant, National Jewish Health

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating Impact of Mycobacterial Physiology on Mycobacteriophage Life Cycles by Mass Spectrometry
Authors: Li Yi , Clase Kari L.
Published: June 2021

Phage‐bacteria interaction plays an important role in both ecology and evolution. Phages lyse bacterial cells to keep nutrient and energy flows of the environment. Arm‐race between phages and bacteria drives evolution of both phages and bacteria. Current phage infection model demonstrates two typical types of phage‐bacteria interaction: lytic and lysogenic cycles. However, the model emphasizes the actions of individual phage particle and bacterial cell. Several researches have unveiled that phage‐bacteria interaction is behavior of both phage and bacterial communities, and a portion of bacterial cells can turn unsusceptible for phage infection. Since phage infection is initiated after the attachment of phage particles to bacterial cells, bacterial surface properties may impact phage‐bacteria interaction. Mycobacterium smegmatis (M. smegmatis) has a thick lipid‐rich cell envelop. Previous researches elucidated that glycopeptidolipids (GPLs) in cell envelope are involved in cell surface properties, receptors of phages and colony morphology of M. smegmatis. Therefore, GPLs may impact phage‐bacteria interaction. Besides GPLs, unique lipids including monomycoloyl trehaloses (MMTs) and triacyl glycerols (TAGs) of M. smegmatis may also impact phage‐bacteria interaction. As morphologies of both M. smegmatis cell and colony change with cell growth phase, composition of envelop lipids (GPLs, MMTs, TAGs etc.) and proteins may change with growth phage, which ultimately lead to alteration of phage‐bacteria interaction. To explore how the mycobacterial physiology affects mycobacteriophage life cycles, the impact of bacterial lipids and proteins composition change on phage and bacterial populations need to be investigated.

Five mycobacteriophages from diverse clusters were selected to infect M. smegmatis cell cultures at early exponential and stationary phases for ten hours. Populations of both phages and bacterial cells were determined over time. After four and ten hours of infection, total proteins and lipids were extracted from subsamples of phage‐bacteria mixture, and further analyzed by mass spectrometry. The overall objective of this project is to figure out how phage life cycle alters when bacterial growth phase changes, and which proteins and lipids may be involved. The findings will provide a better understanding of phage‐host interactions over time and contribute towards novel applications in biotechnology.

Support or Funding Information

Howard Hughes Medical Institute, Science Education Alliance; Polytechnic Institute, Purdue University; Department of Agricultural and Biological Engineering, Purdue University; Biotechnology Innovation and Regulatory Science Center, Purdue University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Examination of Piccolo Splice Variants in Regulated Insulin Secretion from Pancreatic β‐Cells
Authors: Quintana Victoria G. , Salamon Rebecca , Fenster Steven D.
Published: June 2021

Insights regarding the regulated secretion of insulin from pancreatic β‐cells has implications for understanding diabetes and regulation of glucose metabolism. Piccolo is a high‐molecular weight protein and part of a multi‐molecular complex involved in insulin secretion. Interestingly, genetic variants of the Piccolo gene (PCLO) have been modestly associated with increased risk of early‐onset type II diabetes among Pima Indians. Piccolo may regulate insulin secretion through a novel mechanism by serving as a Ca2+ sensor that interacts with L‐type voltage‐dependent Ca2+ channels and other molecules associated with insulin secretory vesicles. The purpose of this study is to examine the role of Piccolo and its splice variants in insulin secretion. Piccolo was originally identified as a protein involved in regulation of neurotransmitter secretion at presynaptic active zones. In neurons, PCLO undergoes extensive alternative splicing. Two major splice variants have been found in both neurons and pancreatic cells: isoform‐1 (I‐1) and isoform‐2 (I‐2). I‐1 has two Ca2+‐binding C2 domains, C2A and C2B, while I‐2 includes only C2A. Alternative splicing also leads to additional minor splice variants with altered Ca2+‐binding properties. Specifically, an exon‐skipping event results in a splice variant that lacks exon 16, a 27‐nucleotide (nt) exon within the C2A domain. Exclusion of this exon (i.e. short variant) enhances the Ca2+‐ and phospholipid binding affinity of C2A motif of rat Piccolo. In this study, we sought to examine the role of Piccolo and its splice variants in Ca2+‐mediated insulin secretion in rat INS‐832/13 pancreatic β‐cells. We demonstrate that INS‐832/13 cells express both I‐1 and I‐2 and also the long and short variant of the C2A domain. In addition, we used CRISPR‐CAS gene editing to selectively target exon 1 of PCLO to examine how loss of function of Piccolo may affect glucose‐stimulated insulin secretion. Finally, we examined the effect of overexpression of the short or long C2A‐domain on insulin secretion.

Support or Funding Information

Research in this study was funded by NIH‐Maximizing Access to Research Careers Undergraduate Student Training in Academic Research (MARC U*STAR) award number T34GM092711, start‐up funds provided to Dr. Steven Fenster (Fort Lewis College), and funds received from SSNAP (Science Scholars the Native American Path) by SACNAS (Dr. Les Sommerville, Fort Lewis College). We would also like to thank Dr. Christopher Newgaard at Duke University for supplying the pancreatic cells and culturing procedure.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Great, Green, Glowing Worms: The Insulin/IGF‐1 Signaling Pathway Regulates C. Elegan Feeding Behavior
Authors: Gousy Nicole , Quadros‐Mennella Princy
Published: June 2021

Type II diabetes mellitus is caused by cellular insulin resistance resulting in increased blood glucose levels. Insulin exerts its actions through a signaling cascade that can be modeled in the Caenorhabditis elegan. The Insulin/IGF‐1 signaling (IIS) pathway found in C. elegans contains the same key genes that are involved in human insulin signaling. These genes include the Daf‐2 gene, encoding for an insulin‐like receptor, the Daf‐16 gene, encoding for transcription factors, and Age‐1 gene, encoding for the phosphatidylinositol 3‐kinase (PI(3)K). When C. elegans sense food in their environment, insulin is released, initiating the IIS pathway, resulting in food uptake via pharyngeal pumping. This study will explore the effects that these specific genes have on feeding behaviors based on how knockout mutations of these genes affect the insulin signaling pathway. To study the pumping rate as in indicator of insulin signaling, we will measure the amount of E. coli in their intestines. The greater the rate of pumping, the higher the concentration of E. coli within the intestines. The worms will be fed GFP‐expressing E. coli so that when placed under a confocal microscope, the E. coli within the intestines will fluoresce. From there the intensity of fluorescence can be analyzed for fluorescence intensity using image analysis software and will be compared between the different mutants. We hypothesize that the Daf‐2 and Age‐1 mutants will show fluorescence indicative of little feeding activity since both of these genes further activate components of the pathway to instigate feeding. However, we hypothesize that the Daf‐16 mutants will show fluorescence similar to that of the wildtype nematodes. This is because Daf‐16 works as a negative inhibitor towards the signaling pathway. This research is significant because it can provide how mutations in homologous genes can contribute to type II diabetes.

Support or Funding Information

We would like to acknowledge the help of Dr. Yadilette Rivera Colon, for her expertise, as well as Emily Burns for her lab assistance. In addition, we would like to thank Eileen Stern for her financial contribution under the Stern Scholarship for Undergraduate Researchers.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Perturbation of FoxO1 expression levels in 3T3‐L1 pre‐adipocytes using cinnamon extract
Authors: Dingess Alexandria , Bova Kaitlin , Aulthouse Amy , Stockert Amy
Published: June 2021

Type 2 diabetes and obesity are among the most prevalent chronic diseases in the United States. Recent research has suggested that these disease states lead to numerous changes in the transcriptome that can be both causal or progressive in nature. Cinnamon has been explored as a potential therapy for type 2 diabetes based on observed decreases in blood glucose in some clinical studies, although its therapeutic benefits are still challenged and its mechanisms of action are still unknown. Aqueous cinnamon extract contains polyphenols, which have been shown to elicit epigenetic changes in several studies. Several key metabolic regulators, thought to be sensitive to epigenetic changes have been identified as players in these disease states. Metabolic regulator FoxO1 is one potential protein sensitive to cinnamon treatment. In addition to metabolic control, FoxO1 has been implemented in numerous other processes including aging, cell death, and lipid localization. Here we present the changes in FoxO1 expression in 3T3‐L1 pre‐adipocytes during early and mid‐stage differentiation both in the presence and absence of cinnamon extract. We have identified increases in FoxO1 expression during mid‐stage differentiation when treated with cinnamon extract. Together with clinical data that demonstrates a phenotype of decreased blood glucose levels in some type 2 diabetic patients when treated with cinnamon, these data suggest the involvement of FoxO1 in the anti‐diabetic mechanisms of cinnamon.

Support or Funding Information

Ohio Northern University Raabe College of Pharmacy Bower, Bennett and Bennett Research Award

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sustained O‐GlcNAcylation Amplifies Erk Signaling
Authors: Cork Gentry K. , Tan Ee Phie , Thompson Jeffrey , McGreal Steven , Machecek Miranda , Kelsh John , Peterson Kenneth , Apte Udayan , Slawson Chad
Published: June 2021

O‐linked N‐Acetylglucosamine, more commonly referred to as O‐GlcNAc, is a single glycosidic post‐translational modification. OGT (O‐GlcNAc Transferase) and OGA (O‐GlcNAcase) are the sole enzymes responsible for the addition (O‐GlcNAcylation) and removal of O‐GlcNAc, respectively. O‐GlcNAc, OGA, and OGT are present in all multicellular organisms and organ tissues. Importantly, OGT knockouts are embryonically lethal, demonstrating the requirement for O‐GlcNAcylation in early organismal development. Like most intracellular post‐translational modifications, O‐GlcNAc regulates a number of cellular pathways including mitochondrial respiration, cell cycle progression, and transcription. Recently, we demonstrated that prolonged treatment of SY5Y neuroblastoma cells with Thiamet‐G (TMG), an OGA inhibitor, caused a dramatic reprogramming of the transcriptome. Therefore, we performed next generation RNA sequencing to investigate the transcriptome changes from isolated liver tissues from mice treated with TMG for two weeks. We also performed the same analysis for liver tissue containing an OGT knockout (OGT‐KO) to compare the effects of sustained O‐GlcNAcylation to the loss of O‐GlcNAcylation. Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed differences in expression for gene sets between the two groups. Further analysis of the data demonstrated an inverse correlation of transcriptional changes for several gene families between TMG‐treated and OGT‐KO. MAPK/ERK signaling genes were among these families, which we found to be of interest. ERK (extracellular regulated protein kinase) phosphorylates a wide variety of protein substrates, regulating processes such as apoptosis, transcription, and cell cycle progression. OGT‐KO was predicted to down‐regulate this pathway while our previous data predicted TMG to up‐regulate the pathway. To test this observation, we grew HeLa cells in TMG for three weeks and serum starved them overnight. The following day, we returned the serum to the media and harvested the lysate at 0, 5,10, 20, 60, and 120 minute time points. When we performed a western blot analysis of the results, the TMG‐treated group demonstrated dramatic amplification of pERK. This observation demonstrates a novel role for O‐GlcNAc as a regulator of the ERK signaling pathway.

Support or Funding Information

NIH NIDDK R01 DK100595

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Combining mass spectrometry and glycan array data to explore invertebrate glycomes
Authors: Wilson Iain , Eckmair Barbara , Hykollari Alba , Yan Shi , Vanbeselaere Jorick , Paschinger Katharina
Published: June 2021

As our glycoanalytical possibilities increase and the amounts of glycomic data on non‐mammalian species accumulate, we can start to consider whether the structures of glycoconjugates from invertebrates can be fitted into a sort of glycophylogeny and which protein receptors recognise them. Our recent data show that N‐glycans from a variety of nematode, mollusc and insect species contain a range of core and antennal modifications with fucose, galactose, glucuronic acid, sulphate, phosphoethanolamine, aminoethylphosphonate and phosphorylcholine. On one hand, we employ an off‐line HPLC/MALDI‐TOF‐MS workflow to assess the actual glycan structures; on the other, beyond previous Western blotting data, we are now printing invertebrate N‐glycomes in a microarray format. Thereby, we are uncovering interactions with different lectins, pentraxins and antisera. This approach should allow us to explore whether and how invertebrate glycomes, especially from medically or biotechnologically relevant species, interact with mammalian immune systems.

Support or Funding Information

This work is funded by the Austrian Science Fund (FWF)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding Heparin/Hparan Sulfate Biosynthetic Pathway in the Generation of Antithrombin Binding Motif using Combinatorial Virtual Library Screening (CVLS)
Authors: Bi Yiling , Sankaranarayanan Nehru Viji , Kuberan Balagurunathan , Desai Umesh R.
Published: June 2021

Heparin/Heparan sulfate glycosaminoglycans (HS‐GAG), is an essential sulfated polysaccharide that regulates multiple biological and pathological processes. Previous biochemical studies have narrowed down antithrombin III binding heparin/heparan sulfate pentasaccharide binding motif responsible for its anticoagulant activity. However, the synthetic sequence did not elucidate how the binding motif is generated in the HS‐GAG biosynthetic pathway. Therefore, novel approaches are required to understand the generation of antithrombin binding domain in the biological system. Here, we utilized computational modeling to understand how different HS biosynthetic enzymes evolve and coordinate together to generate antithrombin binding domain. We constructed a virtual library of tetrasaccharide and hexasaccharide HS sequences with different disaccharide combinations and modifications. Based on 3‐O‐sulfotransferase isoform 1 (3OST‐1) crystal structure and the Combinatorial Virtual Library Screening (CVLS), we found that pentasaccharide could be the minimum recognition motif for 3OST‐1 enzyme. Also, through enzyme‐substrate specificity analysis, we found that 3OST‐1 preferred 6‐O‐sulfo, N‐sulfoglucosamine (GlcNS6S) to N‐sulfoglucosamine (GlcNS), while 2‐O‐sulfation was dispensable, suggesting that 6‐O‐sulfation occur before 3‐O‐sulfation and facilitate 3OST‐1 recognition. In comparison study with 3OST‐3a, which only generates glycoprotein D binding site, we also rationalized the positioning of glucuronic acid (GlcA) instead of the iduronic acid (IdoA) at the non‐reducing end of the modification site. A chemoenzymatic approach was exploited to further validate our findings. Our study used antithrombin domain generation as a test case, and could pave the way to study other GAG biosynthetic enzymes to advance our knowledge of the HS‐GAG biosynthetic pathway.

Support or Funding Information

NIH P01 (HL107152) Programs of Excellence in Glycosciences (PEG)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Beyond ERAD: N‐glycanase will bring you to tears
Authors: Tambe Mitali , Ng Bobby , Freeze Hudson
Published: June 2021

Patients with mutations in NGLY1 cannot make tears (alacrima), have global developmental delay, movement disorder and liver dysfunction. N‐glycanase 1 (NGLY1) de‐glycosylates misfolded N‐glycosylated proteins in the cytoplasm as part of the ERAD pathway prior to their proteasomal degradation. Surprisingly, NGLY1‐deficient patient cells do not accumulate cytoplasmic misfolded N‐glycoproteins, suggesting a more complex function. Interestingly, new studies show NGLY1 is needed for transcription factor NFE2L1/NRF1 activation and normal mitochondrial function.

Using WT and NGLY1‐deficient mouse embryonic fibroblasts (MEFs) we found that NGLY1‐deficient cells were resistant to hypotonic lysis compared to WT. The same was seen in NGLY1‐patient fibroblasts. Additionally, MEFs deficient in both NGLY1 and ENGase (another deglycosylation enzyme) were even more resistant to hypotonic lysis compared to NGLY1‐deficient cells. Water influx and cell swelling precedes cell lysis. We found that NGLY1‐deficient MEFs swell slower than WT MEFs. Since aquaporins (AQP) transport water, we hypothesized that AQP levels might be disrupted in NGLY1‐deficient cells. We found both AQP1 mRNA and protein were reduced in NGLY1‐deficient MEFs. shRNA knockdown of AQP1 in WT MEFs decreased hypotonic lysis, suggesting AQP1 is associated with hypotonic lysis. NGLY1 shRNA and CRISPR studies confirmed that NGLY1 regulates AQP1 levels and hypotonic cell lysis. Preliminary studies show that complementing NGLY1‐deficient MEFs with NGLY1 increases AQP1 and restores hypotonic lysis. Current efforts are directed towards understanding whether NGLY1 enzyme activity is necessary for AQP regulation. 13 types of AQP are expressed in different cell types. Our preliminary data suggest that AQP11 levels are disrupted in NGLY1‐deficient patient cells and CRISPR cells, suggesting that NGLY1 might regulate different types of AQP.

We have identified a novel function of NGLY1, i.e. to directly/indirectly regulate AQPs. This finding may relate to NGLY1‐deficient patients' inability to make tears. Future efforts will try to identify NGLY1‐dependent transcription factors responsible for regulating AQPs. This work is supported by the Bertrand Might Research Fund and NGLY1.org.

Support or Funding Information

This work is supported by the Bertrand Might Research Fund.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anti‐aging effect and gene expression profiling of Bumblebee (Bombus terrestris) queen glycosaminoglycan in aged rats
Authors: Ahn Mi Young , Yun Hyung Joo , Hwang Jae Sam
Published: June 2021

Glycosaminoglycans have been used to diminish deleterious effects associated with aging by preventing destruction of cartilage, bone, discs, and skin. The objective of this study was to evaluate the anti‐aging effect of a newly prepared glycosaminoglycan (GAG) derived from bumblebee (Bombus terrestris) queen (BtQG, 10 mg/kg). Gryllus bimaculatus (Gb, cricket) GAG (GbG, 10 mg/kg) or glucosamine sulfate (25 mg/kg) was used as a positive control. They were intraperitoneally administered to old SD rats as part of their diets for one month. BtQG reduced serum levels of creatinine and BUN. It possessed anti‐lipidemic activities, hepato‐and renal‐protective actions, and maintained normal glucose level in treated rats. BtQG and GbG had marked anti‐inflammatory effects. They inhibited free fatty acid, ALP, and sGPT values. In addition, they showed anticoagulant and antithrombotic effects. The concentration of factor 1 (fibrinogen) was increased in BtQG treated rat plasma. In microarray study, CaG5‐treated rat group shows upregulation of 36 genes compared to the control group, including secretogranin II (Scg2), AP‐1‐regulated protein related ROS DNA damage repair, metallothionein 1a, metallothionein 1M, and alpha‐2 macroglobulin. It also showed 417 downregulated genes, including vimentin, moesin, an d mitochondrial carbonic anhydrase.

Support or Funding Information

This work was supported by the Rural Development Administration Basic Research project, PJ011853

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hybrid biosensing cellulose‐based scaffolds for imaging‐assisted tissue engineering
Authors: O'Donnell Neil , Okkelman Irina A. , Ruane Shannen A. , Timashev Peter , Gromovykh Tatyana I. , Dmitriev Ruslan I.
Published: June 2021

3D tissue models closely mimic in vivo cell and tissue organization otherwise not seen in conventional cell culture. However, live cell imaging and quantification of cells growing within 3D scaffold are yet to be achieved for success of tissue engineering, cancer biology and regenerative medicine. Here, we aimed to design the environment‐sensitive scaffold material acting both as supporting material and a biosensor for cells growing in it. We chose cellulose‐binding domain of C. fimi CenA protein, termed as CBD (N‐terminal fragment, Ala32‐Thr137) and genetically fused it to either pH (enhanced cyan fluorescent protein, ECFP) or Ca2+ (gCaMP2)‐sensitive proteins. ECFP and gCaMP2 are prospective biosensors for quantitative Fluorescence Lifetime Imaging Microscopy (FLIM). Next, we optimised E. coli expression, purification and folding conditions for these chimeric proteins, achieving yields of ~6 mg/L culture, bright fluorescence (~55% folding rate) and sensitivity to extracellular pH and Ca2+.

To assemble the functional biosensing scaffolds using CBD‐fusions, we performed labeling of the three different types of cellulose‐based materials: commercially available nanofibrillar cellulose Growdex™, bacterial cellulose spheres and decellularized plant tissues. Both CBD‐ECFP (pH‐sensitive) and CBD‐gCaMP2 (Ca2+‐sensitive) proteins efficiently labeled scaffolds in concentration‐dependent manner and remained stable under physiological conditions for > 7 days. In a proof‐of‐concept experiments, we tested usability of CBD‐ECFP‐stained Growdex scaffold as an extracellular pH sensor in FLIM: we observed robust and reversible changes in fluorescence lifetime (2.3–2.5 ns) over the range of physiological pH. Further, we tested the biosensing properties of CBD‐ECFP scaffolds, populated with cultured human colon cancer HCT116, mouse embryonic fibroblast (MEF) and primary stem cell‐derived mouse intestinal organoids. All the types of scaffolds were compatible with growth of cells with no evident toxicity, confirmed by experiments with unlabelled scaffolds.

In conclusion, we have developed versatile labeling approach for producing biosensing cellulose‐based 3D scaffolds, suitable for different biosensors and types of cell and tissues, including single cells, spheroids and organoids. Such ‘hybrid’ biosensing scaffolds allow for real‐time measurement of extracellular biomolecules using quantitative FLIM microscopy, providing improvements in multi‐parametric imaging of engineered tissue models.

Support or Funding Information

Science Foundation Ireland grant SFI 13/SIRG/2144.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exosomal miRNAs derived from specific cardiac progenitor cells exert strong therapeutic effect on myocardial infarction
Authors: Xuan Wanling , Wang Lei , Tang Yaoliang , Ashraf Muhammad
Published: June 2021

Objective

miRNA signatures are unique in exosomes amongst different parent cells. Here, we isolated and characterized exosomes from small molecule induced cardiac progenitor cells (Exo‐CPCiso) and determined their effects on cardiac cells in vitro and on mice hearts following myocardial infarction (MI).

Methods and results

miR‐373, miR‐367, miR‐520 and miR‐548 were significantly overexpressed in Exo‐CPCiso and these miRNA microarray data were confirmed with real‐time PCR. Go enrichment analysis based on miRNA‐targeted genes showed that biological process included organelle, molecular function, response to stress and mitotic cell cycle. Interestingly, expression of fibrotic genes in cultured fibroblasts stimulated with TGF‐β was significantly downregulated after treatment with Exo‐CPCiso. Loss‐of‐function analysis using miR‐373 inhibitor confirmed that enrichment of miR‐373 was the major contributor to reversion of TGF‐β stimulation in fibroblasts. Moreover, intramyocardial injection of Exo‐CPCiso exerted favorable cardioprotective effects on cardiomyocyte proliferation, angiogenesis and preservation of cardiac function in mice one month post‐MI compared with PBS and Exo‐iPSC treated mice.

Conclusion

Exosomes derived from cardiogenic small molecule induced‐CPCs were highly effective in myocardial repair and functional improvement after MI due to their unique miRNA cargo contents. These data further suggest that cellular source of exosomes has a strong bearing on their therapeutic potential.

Support or Funding Information

This study was supported by the National Institutes of Health grants, RO1 HL126516, HL134354, 2RHL086555 and RO1 AR070029.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TIMP‐2 Inhibits Triple Negative Breast Cancer Growth and Metastasis through EMT Suppression and Promotion of Vascular Normalization
Authors: Jensen Sandra M. , Kumar Sarvesh , Chowdhury Ananda , Castro Nadia , Shih Joanna , Salomon David S. , Stetler‐Stevenson William G.
Published: June 2021

Successful tumor cell invasion requires degradation of the extracellular matrix (ECM) by matrix metalloproteases (MMPs), a large family of metalloproteinases that cleave ECM proteins. MMP synthesis and activation is tightly regulated by their natural inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). TIMP‐2 is a multifunctional protein shown to inhibit tumor growth, invasion and metastasis independent of MMP inhibitory activity via mechanisms including alteration of the epithelial to mesenchymal transition (EMT), reduced tumor angiogenesis, and inhibition of VEGF‐A induced vascular permeability. Triple‐negative breast cancers (TNBCs) are highly vascular tumors that exhibit EMT plasticity and extensive metastatic dissemination. We explored the efficacy of recombinant TIMP‐2 (rTIMP‐2) as a preclinical therapeutic using an orthotopic mouse model which phenotypically resembles human TNBC and establishes spontaneous lung metastases. Daily rTIMP‐2 treatment (100 ug/kg/day) was initiated 10 days post tumor cell implantation and continued through day 37. Biweekly tumor measurements [tumor volume) = 1/2(length × width2)] revealed reduced primary tumor growth in rTIMP‐2 treated mice 15 days post inoculation and maximum tumor growth inhibition on day 29. Immunostaining of primary tumors demonstrated increased CK‐18 expression along with decreased CK‐14 and vimentin in rTIMP‐2 treated mice, suggesting reversal of the EMT phenotype by day 32, while lung histology showed a significant reduction in metastatic foci in rTIMP‐2 treated mice at day 52. Moreover, immunofluorescence demonstrates increased pericyte coverage and reduced vascular leakage in rTIMP‐2 treated primary TNBC tumors, showing modulation of vessel morphology and promotion of events consistent with vessel normalization. These findings suggest the therapeutic potential for rTIMP‐2 in the clinical treatment of TNBC.

Support or Funding Information

This work was funded by the NCI Center for Cancer Research Intramural Research Program Project # ZIA SC009179‐25.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dopamine Transporter Allosteric Modulator SRI‐32743 Alters The Effects Of Cocaine On Dopamine Neurotransmission In Adult Mice
Authors: Hager Audrey , Daws Lynette , Ananthan Sam , Beckstead Michael
Published: June 2021

Cocaine is a highly abused psychostimulant that inhibits dopamine (DA) uptake by blockade of the dopamine transporter (DAT). Despite knowledge and understanding of cocaine's main mechanism of action, there are no FDA‐approved pharmacotherapies for treatment of cocaine‐use disorder. Recently novel allosteric modulators of DAT have been identified that have nanomolar potency to partially inhibit DAT uptake, which may be useful as probes of biogenic amine transporter function and may have therapeutic potential. Here we used both patch clamp electrophysiology in male and female mouse brain slices and high speed chronoamperometry in anesthetized mice to examine the effects of two novel ligands (SRI‐32743 and SRI‐35282) on dopamine neurotransmission and their ability to alter the effects of cocaine. Pretreatment with SRI‐32743 blunted the cellular effects of cocaine by altering DAT function in a concentration‐dependent manner. In addition, SRI‐32743 decreased D2 receptor‐mediated currents of midbrain DA neurons. In contrast, the ligand SRI‐35282 demonstrated less ability to reduce the effects of cocaine. These preliminary results are promising and further exploration of the effects of these allosteric DAT modulators on behavior will be important for determining their potential as pharmacotherapies for the treatment of cocaine‐use disorder.

Support or Funding Information

R01DA32701, T32DA031115, F32DA042569, R01MH106978

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reinforcing Potency and Effectiveness of Synthetic Cathinones: Potency versus Selectivity for DAT
Authors: Gannon Brenda M. , Sulima Agnieszka , Rice Kenner C. , Baumann Michael H. , Collins Gregory T.
Published: June 2021

Synthetic cathinones found in “bath salts” preparations interact with monoamine transporters and function as either cocaine‐like monoamine uptake inhibitors or amphetamine‐like substrates. Abuse of these synthetic cathinones has resulted in regulation of some cathinones internationally. Modifications to the chemical structure of regulated cathinones (e.g., MDPV) produce a series of new cathinones (e.g., α‐PVP, α‐PPP, MDPBP, and MDPPP) which may not be subject to existing regulations. Although MDPV is a more potent and effective reinforcer than cocaine, little is known about the reinforcing effects of other synthetic cathinones. Thus, this study examined intravenous self‐administration of cocaine, MDPV, α‐PVP, α‐PPP, MDPBP, and MDPPP to test the hypotheses that 1) stimulants with greater potency to inhibit the dopamine transporter (DAT) are more potent reinforcers, and 2) stimulants with greater selectivity for DAT relative to the serotonin transporter (SERT) are more effective reinforcers. Potencies to inhibit monoamine uptake at DAT and SERT were determined using synaptosomes prepared from rat brains. Separate groups of male Sprague‐Dawley rats (n=12 per group) were used to generate full dose‐response curves under a progressive ratio (PR) schedule of reinforcement for α‐PVP, α‐PPP, MDPBP, and MDPPP; full dose‐response curves for MDPV and cocaine were also generated in each rat (n=48). Rank order potency to inhibit DAT was MDPV > α‐PVP ≈ MDPBP > MDPPP ≈ α‐PPP ≈ cocaine, while rank order DAT/SERT selectivity was α‐PVP > α‐PPP > MDPV > MDPBP > MDPPP > cocaine. The potency to inhibit DAT correlated with the potency of each drug to maintain responding (MDPV > α‐PVP ≈ MDPBP > MDPPP ≈ α‐PPP ≈ cocaine). Relative reinforcing effectiveness as determined by comparing the maximum number of infusions obtained for each drug under a PR schedule of reinforcement was α‐PVP > α‐PPP ≈ MDPV > MDPBP ≈ MDPPP > cocaine, which correlated with DAT/SERT selectivity. These data suggest that (1) potency at DAT is an important determinant of the potency of each drug to function as a reinforcer, (2) each of these synthetic cathinones functions as a more effective reinforcer than cocaine, and (3) DAT/SERT selectivity is related to reinforcing effectiveness and may predict the abuse‐related effects of novel synthetic cathinones in humans.

Support or Funding Information

These experiments were supported by NIH grants R01DA039146 (GTC) and T32DA031115 (BMG) and the Intramural Research Programs of NIDA (MHB, KCR) and NIAAA (KCR).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sex Differences in High Fat Diet‐Induced Enhanced Sensitivity to the Locomotor Stimulating Effects of Methamphetamine and SKF 82958
Authors: Ramos Jeremiah , Robles Grace Flores , Gonzalez Adrian , Serafine Katherine
Published: June 2021

Eating a high fat diet can impact dopamine systems; the same systems that are targeted by drugs of abuse. Rats eating high fat laboratory chow are more sensitive to the behavioral effects of abused drugs, including methamphetamine. Most of the previous research investigating diet‐induced effects on drug sensitivity has used male rats, therefore it is not known if females eating high fat chow are also more sensitive to the behavioral (e.g., locomotor‐stimulating) effects of methamphetamine. Methamphetamine‐induced locomotion is mediated, in part, by dopamine D1 and D2 receptors. While several reports have examined diet‐induced effects on dopamine D2 receptor agonists, it is not known if eating high fat chow also enhances sensitivity of rats to dopamine D1 receptor agonists (e.g., SKF 82958). To test the hypothesis that eating high fat chow enhances sensitivity of male and female rats to drugs that act on dopamine systems, male and female Sprague‐Dawley rats eating either standard laboratory chow (17% kcal from fat) or high fat chow (60% kcal from fat) were tested once per week for 6 total weeks with SKF 82958 (0.01–3.2 mg/kg) or methamphetamine (0.1–3.2 mg/kg) using a cumulative dosing procedure. In an initial cohort of animals (n=3/group), eating high fat chow tended to enhance sensitivity of female rats to SKF 82958 and methamphetamine. This trend was less apparent among male rats eating different diets. Consistent with previous reports, females tended to be more sensitive than males to the behavioral effects of both drugs, regardless of diet. Taken together with previous reports, the present study demonstrates that robust sex differences regarding the behavioral effects of dopaminergic drugs warrant further investigation of both sexes to understand the impact of diet on drug sensitivity. Future research will examine the effects of diet on sensitivity to the reinforcing effects of methamphetamine, to further assess how diet might impact abuse vulnerability.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of hyperbaric oxygen (HBO2) as a treatment for hyperalgesia associated with naloxone‐precipitated withdrawal in morphine‐dependent mice
Authors: Brewer Abigail Lucille , Shirachi Donald Y. , Quock Raymond M.
Published: June 2021

Opioid dependence has become a major problem in the United States. Withdrawal from opioid drugs is associated with hyperalgesia (increased sensitivity to a pain stimulus) which may contribute to relapse [Carcoba et al., J Addict Dis 30(3): 258–70, 2012]. Hyperbaric oxygen (HBO2) is 100% oxygen at higher than atmospheric pressure. HBO2 has recently been shown to be effective at decreasing physical signs of withdrawal in morphine‐dependent mice, and there is a growing body of research showing that HBO2 is effective at decreasing pain in clinical conditions and in preclinical modeling [Nicoara et al., Brain Res. 1648:434–7, 2016; Efrati et al., PLOS One 10(5)1–25, 2015; Gibbons at al., Brain Res. 1537:111–6, 2013]. The effectiveness of HBO2 to decrease hyperalgesia caused by opioid withdrawal in an animal model of drug dependence was evaluated in this study. Male and female NIH Swiss mice were made dependent upon morphine by injecting animals s.c. with twice‐daily escalating doses of morphine for 4 consecutive days. On the 5th day, animals were given a final injection of the highest dose of morphine and injected i.p. with 1.0 mg/kg naloxone 8 hours after the final morphine injection. Animals were treated with 30 min of HBO2 at 3.5 atmospheres absolute or room air 60 min prior to naloxone injection. Nociception was evaluated using the tail withdrawal, hot plate and paw pressure tests every 10 min for 30 min after naloxone injection. Baseline levels of nociception for each endpoint were taken prior to induction of morphine dependence. Male and female animals exhibited hyperalgesia in the hot plate and paw pressure tests but not the tail withdrawal test. HBO2 significantly decreased the amount of hyperalgesia in the hot plate and paw pressure tests in male animals. In female animals, HBO2 had no effect on hyperalgesia in the hot plate test but significantly decreased the level of hyperalgesia associated with the paw pressure test. While no hyperalgesia was seen in the tail withdrawal test, HBO2 did produce significant antinociception in male and female animals. These intriguing results show that HBO2 is capable of reducing hyperalgesia associated with opioid withdrawal and warrant further investigation. Furthermore, these results help support previous findings showing that HBO2 may be effective at decreasing symptoms of opioid withdrawal.

Support or Funding Information

This research was supported by the Marchionne 12‐month Fellowship from the Department of Psychology at Washington State University, NIH Grant AT‐007222, and the Honors College Distinguished Professorship from Washington State University.

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