bluuebunny/crossref_metadata_2025 huggingface dataset:
Copper distribution networks in Pseudomonas aeruginosa
Authors: Arguello Jose , Quintana Julia , Novoa‐Aponte Lorena
Published: June 2021

Bacterial Cu+ homeostasis is based on protein networks that mobilize copper to cellular targets, enabling both metallation of cuproproteins and maintenance of non‐toxic metal levels. Here, we use genome wide expression analysis (RNA‐Seq) to characterize the response of Pseudomonas aeruginosa to external 0.5 mM CuSO4, a condition where cell viability is uncompromised. Analysis of copR and cueR mutant strains revealed that compartmental Cu levels are independently controlled; the cytoplasmic Cu sensor CueR regulates cytoplasmic chaperones and plasma membrane transporters, whereas the two component system CopR/S shows a putative DNA recognition sequence, forming a regulon composed of genes involved in periplasmic Cu‐homeostasis. Response to Cu‐imbalance resulted in distinct transcriptomic landscapes: a pre‐steady‐state (5 min), where general metabolism is slowed down, and a steady‐state (2 h), where general metabolism is restored. Specific Cu homeostasis genes were strongly regulated in both conditions. Our system‐wide analysis revealed induction of three Cu‐efflux systems (a P1B‐ATPase, a porin, and a resistance‐nodulation‐division (RND) system), a putative Cu‐binding periplasmic chaperone and the unusual presence of two cytoplasmic CopZ proteins. Importantly, novel transmembrane transporters probably mediating Cu‐influx were among those largely repressed genes upon Cu‐stress. Three proteins were identified among these: OprC, an outer membrane porin; PA3789, an inner membrane protein; and PA5030, a Met/His rich protein, member of the major facilitator superfamily (MFS) of transporters. Alternatively, genes encoding previously proposed Cu‐importers ccoA and hmtA did not change their expression levels. The occurrence of cell influx transporters is a prerequisite for homeostasis of a given cellular constituent. However, deletion of the putative Cu‐importers OprC and PA1530 does not affect the Cu content of whole cells, suggesting that Pseudomonas has alternative ways to acquire Cu. As Pseudomonas secrete two major siderophores (pyochelin and pyoverdin) to scavenge metals, we hypothesize that those siderophores will constitute an alternative way to import copper. Interestingly, genes involved in both, pyochelin and pyoverdin pathways, are overexpressed when the outer membrane porin is absent, but only pyochelin‐related genes are up‐regulated when the putative inner‐membrane transporters are deleted. This suggests that pyochelin can be the alternative Cu‐import system that Pseudomonas uses under copper‐starving conditions. Summarizing, our studies show a system‐wide network of sensors/regulators, soluble chaperones, and influx/efflux transporters that control the Cu levels in P. aeruginosa compartments.

Support or Funding Information

This work was supported by NIH Grant R01 GM114949 to J.M.A.

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 cytoplasmic Cu+‐chaperones of Pseudomonas aeruginosa
Authors: Novoa‐Aponte Lorena , Arguello Jose M.
Published: June 2021

Copper is a micronutrient required by multiple enzymes as a redox co‐factor. However, free Cu is toxic, leading to Fe‐S cluster disassembly and reactive oxygen species. As such, immune cells use phagosomal Cu‐overload to clear pathogens. Thus, bacteria need to direct Cu to correct targets while maintaining non‐toxic levels. To limit toxicity, cells sequester and drive the efflux of Cu using various mechanisms. Among these, single cytoplasmic Cu‐chaperones mobilize the metal within the cell compartments. We recently used RNA‐seq to show that Pseudomonas has a network of proteins that mobilize Cu among cellular compartments. Importantly, our study uncovered the unusual presence of two putative Cu‐chaperones in these bacteria (CopZ1 and CopZ2). Both are under the control of the cytoplasmic Cu‐sensor CueR and consequently copZ1 and copZ2 expression is induced by exposure to Cu. Why is there two Cu‐chaperones in Pseudomonas? and why do both respond to Cu‐imbalances? We hypothesize that metabolically linked via Cu‐exchange, these two Cu‐chaperones deliver metal to different targets. We have observed that CopZ1 and CopZ2 share 37% identity (51% similarity), contain the invariant Cu‐binding motif (CxxC), and exhibit similar secondary structure in line with the βαββαβ‐fold, characteristic of Cu‐chaperones. Both proteins bind Cu with 1:1 stoichiometry and sub‐femtoMolar affinities. However, CopZ2 has 100‐times higher affinity for Cu compared to that of CopZ1. We determined the exchange of Cu between both proteins. As expected from the affinity constants, the Cu‐exchange equilibrium is displaced to the formation of the metallated‐CopZ2 form. Interestingly, both chaperones are able to transfer Cu to non‐cognate chaperones from other organisms, suggesting limited molecular recognition during metal‐exchange. Although both genes are equally induced by Cu‐stress, we observed that CopZ2 is 10‐times more abundant than CopZ1, under resting conditions and upon Cu stress. The differential response to Cu (affinities), protein levels, and their distinct electrostatic surface potential suggest that CopZ1 and CopZ2 deliver Cu to different targets and perhaps receive the metal from different molecules. Currently, we are testing which are these targets and Cu‐donors.

Support or Funding Information

This work was supported by NIH Grant R01 GM114949 to J.M.A..

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Integrative analysis of sex differences in adipose tissue gene expression
Authors: Anderson Warren , Guertin Michael , Civelek Mete
Published: June 2021

Sex differences in adipose tissue distribution and function are associated with cardiometabolic disease risk. While extensive research efforts have revealed sex differences in cell signaling and physiology, there is a relative dearth of information regarding the contribution of transcriptional regulation to sex differences in adipose function. We investigated sex‐dependent features of transcriptional regulation in subcutaneous adipose tissues using multiple publicly available omic‐scale gene expression data sets from geographically and ethnically diverse healthy human populations (N = 111–404 samples). Gene set enrichment analyses revealed that fatty acid metabolism, glycolysis, oxidative phosphorylation, and reactive oxygen species production pathways were enriched for differentially expressed genes (FDR < 0.1). We further identified a core set of 79 genes that were consistently differentially expressed in subcutaneous adipose tissue, but not other metabolic tissues (e.g., ELOVL3, FADS1, RORB, FDR < 0.05). A preponderance of these sex‐dependent genes exhibited expression correlations with metabolic traits that were measured in parallel with genes expression (e.g., LDL cholesterol, blood pressure, and body mass index). The genes that were positively correlated with deleterious metabolic traits were enriched for upregulation in males. Many of these results from humans were replicated in adipose tissue transcriptomic data from a population of 97 genetically diverse mouse strains. To further interrogate the molecular causes and consequences of sex‐dependent gene expression differences, we employed bioinformatic and network inference analyses. We identified putative upstream regulators of gene expression sex differences including PPARG and CEBPB. We identified subtle differences in gene expression coordination based on network analysis. A related but distinct aspect of sex differences in adipose function is that there are sex differences in the associations between genetic variants and body fat distribution‐related traits. Current efforts are underway to determine whether these sex‐dependent influences of genetics on adipose distribution could be mediated by influences on gene expression in adipose tissue, as suggested in the literature but not previously demonstrated. Our findings and related investigations have the potential to impact precision medicine approaches that account for the influences of sex on the development and perpetuation of cardiometabolic diseases.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel Rac Inhibitors as Targeted Therapeutics for Metastatic Breast Cancer
Authors: Asencio‐Torres Gabriela , Hernández Eliud , Vlaar Cornelis , Dharmawardhane Suranganie , Castillo‐Pichardo Linette
Published: June 2021

Metastatic disease is the primary cause of breast cancer mortality, due to the lack of effective therapy. The Rho GTPase Rac is integral for the promotion of cancer cell migration/invasion, proliferation, and survival. Since metastatic breast cancers often overexpress or exhibit high Rac activity, inhibition of Rac is a viable strategy against metastatic cancer. Recently, we characterized EHop‐016, a small molecule that inhibits Rac activity of metastatic breast cancer cells with an IC50 of 1 μM. EHop‐016, which is 10–100 times more active than previously available Rac inhibitors, inhibits the activity of the Rac downstream effector p21 activated kinase (PAK), lamellipodia extension, and cell migration of metastatic breast cancer cells. Moreover, EHop‐016 at ≥ 25 mg/kg Body Weight (BW) significantly reduced tumor growth, metastasis, and angiogenesis in a mouse model. However, our recent pharmacokinetic study of EHop‐016 in a mouse model demonstrated that its bioavailability should to be improved for further pharmacological development. Our hypothesis is that improvement of the EHop‐016 structure will provide probes with increased potency against Rac and improved bioavailability. Herein we have tested several Ehop‐016 derivatives for their effects on breast cancer cell viability, cell migration, and Rac activation. Using MTT assays we found that the Ehop‐016 derivatives, HV‐107 and HV‐118, significantly inhibit the viability of metastatic breast cancer cell lines MDAMB‐231 and MDA‐MB‐435; while presenting minimal toxicity towards non‐cancerous mammary epithelial cells. The effects of HV‐107 and HV‐118 on the inhibition of Rac activation were tested by ELISA‐based Rac activity assays and pulldown assays. Results show that at 250 nM, HV‐107 inhibits Rac activation by 55%, whereas HV‐118 has a similar effect at 100 nM in MDA‐MB‐231 and MDA‐MB‐435 cells. Moreover, using transwell assays we studied the effects of both Rac inhibitors in metastatic breast cancer cell migration and observed a significant reduction. Finally, we tested HV‐107 in a mouse model of metastatic breast cancer and found a 25% reduction in tumor growth in mice treated with 5 mg/kg BW HV‐107. Taken together, our findings suggest HV‐107 and HV‐118 as promising Ehop‐016 derivatives with potential as anti‐metastatic agents, that should be further characterized.

Support or Funding Information

This study was supported by awards from the Susan Komen for the Cure, NIH/NIMHHD U54MD008149, and the Puerto Rico Science and Technology Trust to SD; NIH/NCRR R25GM061838 to UPR MSC; NIH/NIMHHD RCMI 8G12MD007583RCMI, Title V PPOHA 031M10505 and Title V Cooperative P031S130068 from U.S. Department of Education to UCC; and PRINBRE (NIH/NIGMS P20GM103475‐13) Sub‐Award to LCP.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Extracellular IGFBP‐3 by Humanin in A549 Cells
Authors: Muterspaugh Robert Dean , Price Deanna , Guthrie Jeffrey , Heyl Deborah , Evans Hedeel
Published: June 2021

The insulin‐like growth factor binding protein 3 (IGFBP3) is a multifunctional protein that belongs to a family of IGF binding proteins with highly conserved structures. Previously, an inverse relationship was demonstrated between serum or plasma levels of the protein and lung cancer risk. The protein was found to inhibit the growth and survival of non‐small cell lung cancer (NSCLC) A549 cells and its overexpression potently induced apoptosis in these cells. Humanin is a small mitochondrial‐derived peptide known to bind with high affinity to residues 242–259 of IGFBP‐3. Growing evidence suggests that, when secreted, humanin is a potent cytoprotective peptide against a range of disease models and cellular stress. The role that humanin plays in tumorigenesis and metastasis is not clear in A549 cells.

While both IGFBP‐3 and humanin are known to exert their function both intracellularly and extracellularly, little is known about their extracellular interaction and regulation of A549 cell survival.

Our hypothesis is that humanin acts as an IGFBP‐3 sponge in the extracellular compartment and thereby regulates cell growth and survival in a time and concentration dependent manner. Using peptide synthesis, ELISA, immunoprecipitation, Western blotting, and tissue culture, we examine the physical and functional interaction of extracellular IGFBP3 and humanin both in vitro and in vivo in A549 cells.

Support or Funding Information

This work was supported by an Eastern Michigan University Provost Research Support Award/Chemistry Seller's 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.
Expression and Regulation of CHP and NHE in Cancer
Authors: Bakker Clare , Wallert Mark , Provost Joseph
Published: June 2021

The hypoxic and serum starved environment of metastasizing cancer cells requires a mechanism to overcome the low intracellular pH induced by the Warburg effect. The sodium hydrogen exchanger isoform 1 (NHE1) is a highly regulated, transmembrane protein that modulates pH of extra and intra cellular environment. One set of proteins which bind and regulate NHE1 include calcineurin homologous proteins 1 and 2 (CHP1&2). CHP1 is necessary for basal NHE1 transport while CHP2, increases the exchange activity and Vmax of NHE1. Both proteins bind to nearly the same NHE1 residues however these interactions are not clearly known. To understand the role that all three proteins may play in regulating cellular function, tissue expressional distribution was conducted on tumor and non‐diseased human tissues. Analysis of RNA sequence from Expression Atlas database revealed that different from published record, CHP2 is expressed within a number non‐diseased tissue including but not limited to: spinal cord, urinary bladder, vagina, tibial nerve, lower leg skin. Analysis of RNA sequence databases of cancerous tissue showed significantly higher expressed CHP2 within a number of, but not all cancer tissues including lung, cervical, and esophogeal cancers. Surprisingly prostate and colorectal adenocarcinoma express CHP2 with a 3–10 fold decrease in expression compared to non‐diseased tissue. To further analyse the expression of CHP2 and NHE1 in lung cancer cells, we performed qPCR on human tissue samples. Cq values displayed a significantly higher expression of CHP2 within squamous cell carcinoma, but not adenocarcinoma, in comparison to adjacent healthy tissue. RNAseq, Expression Atlas, analysis of CHP2 within non‐small cell lung cancer also demonstrates this 15‐fold increased expression for squamous cell carcinoma. This signals an integral role of CHP2 for the survival of cancer cells, and hints at the subsequent impact upon the regulation of NHE1 to allow cells to survive nascent tumour acidification. To examine this possibility, we measured the impact of pHi in several cancer cell lines treated with an NHE1 specific inhibitor or after NHE1 or CHP2 knockdown. This work shows that CHP2 expression is required for cells to maintain sustainable pHi for survival. The impact of NHE1 in hypoxic conditions several tumor cell lines. Thus, the increase in expression and change in transport kinetics demonstrates the effect of CHP2 upon proton transport for NHE1, recovering pHi and increasing cell survival.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of mTORC2 and Nitric oxide in Bladder Cancer Invasion
Authors: Sahu Divya , Klemke Richard , Boss Gerry R. , Hansel Donna E.
Published: June 2021

Background

Despite accounting for around 17,000 deaths and 79,000 new diagnoses annually in the United States, treatment options for bladder cancer remain limited. The key driver of bladder cancer stage progression is increasing invasion into the bladder wall, which has limited response to current chemotherapy options and hence newer therapies directed against invasion are key to improving the treatment of bladder cancer. We have recently shown that arginine, the nitric oxide (NO) precursor, is implicated in bladder cancer progression. Our laboratory has also identified mammalian target of rapamycin complex 2 (mTORC2) as a driver of bladder cancer cell invasion. We evaluated the interaction between these two pathways to promote invasion.

Methods

Expression of inducible and endothelial nitric oxide synthases (iNOS and eNOS) in FFPE tissue sections of progressive disease was assessed by IHC and correlated with histopathology, progression and stage based on moderate to strong expression (2+, 3+) compared to absent to weak expression (0, 1+). We utilized gene silencing methods and NOS inhibitors and NO scavenger for effects on bladder cancer invasion and migration. We assessed mTORC2 pathway activity and NOS levels in invasive cell tip protrusions called “invadopodia” and evaluated if mTORC2 regulates NOS localization. We used a novel zebrafish model to characterize the effects of mTORC2 and NO on bladder cancer metastases.

Result

We found that eNOS and iNOS are elevated in invasive human bladder tumors and cell lines and their ablation reduces bladder cancer cell migration and invasion by reorganizing the actin cytoskeleton and reducing invadopodia. mTORC2 silencing can affect levels of iNOS and eNOS in bladder cancer cell lines. Silencing of mTORC2, eNOS or iNOS reduced metastases of bladder cancer cells within zebrafish.

Conclusion

mTORC2 pathway is a key driver of bladder cancer invasion and metastases by regulating the NO pathway and both mTORC2 and NO can be targets for bladder cancer therapy, which would benefit patient outcomes.

Support or Funding Information

Prevent Cancer Foundation Fellowship to DS

Case Western Reserve University/Cleveland Clinic CTSA Grant Number UL1 RR024989 from the National Center for Research Resources (NCRR), a KL2 career development award (RR024990) and UCSD start‐up funding to DEH

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Growth inhibition of breast cancer by two flexible heteroarotinoid enantiomers
Authors: Ginn Emily , Baek Jenny , Zou Hongye , Fallatah Maryam M.J. , Cayton Erica , Liu Shengquan , Louie Maggie
Published: June 2021

Flexible heteroarotinoid (flex‐hets) are compounds that show promising anti‐cancerous activities. More specifically, SHetA2, a first generation Flex‐Het, has been shown to block the growth of cervical, head and neck, kidney, lung, and ovarian cancers, and most recently, prostate and breast cancers. A second generation of compounds was developed to improve SHetA2's high lipophilicity, limited selectivity, low bioavailability, and its complicated synthesis. Results from our recent study suggest that one of these analogs, 1‐(1‐(naphthalen‐1‐yl)ethyl)‐3‐(4‐nitrophenyl)thiourea (SL‐1‐09) exhibits anti‐cancer activities against both ERα+ and ERα‐breast cancer cells at micromolar concentrations. Since SL‐1‐09 is a mixture of enantiomers, R (SL‐1‐29) and S (SL‐1‐30), each analog was synthesized and purified for further analysis. Our results suggest that SL‐1‐30 shows a greater growth inhibitory effect than SL‐1‐29 against both ERα+ (T47D) and ERα‐(MDA‐MB‐453) breast cancer cells with GI50's of 4.56 ±1.17 μM and 3.03 ±0.86 μM, suggesting that the activity observed in SL‐1‐09 is likely associated with the S enantiomer. Furthermore, preliminary cell cycle analysis suggests the percent of MDA‐MB‐453 cells in S‐phase is reduced by 25% when treated with 5.0 μM SL‐1‐30 for 24 hours and 20% reduced when treated with 5.0 μM SL‐1‐30 for 48 hours. Consistent with this data, both T47D and MDA‐MB‐453 express lower levels of cell cycle proteins and genes—cyclin A, cyclin B1, cyclin D1, cyclin E and CDK2 48 hours after treatment with SL‐1‐09 and SL‐1‐30, with cyclin E and CDK2 showing significant changes as early as 12 hours in cells treated with SL‐1‐30. In summary, these results demonstrate that both SL‐1‐30 and SL‐1‐09 inhibit ERa+ and ERa− breast cancer cell growth, potentially by blocking cell cycle progression; however further studies are necessary to delineate the mechanism of action.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Real Time PCR analysis of Ewing's Sarcoma Cell Lines in Response to Chemotherapeutic Treatment.
Authors: Pitzen Sam , Kruchten Anne
Published: June 2021

Ewing's Sarcoma is a rare bone and soft tissue cancer typically found in children ages ten to twenty years old. Survival rate is about 70% if caught before metastasis, but drops to 30% if the cancer has metastasized throughout the body. Treatment options for Ewing's Sarcoma include amputation, radiation, and/or chemotherapy. To better understand the effects of chemotherapeutic drugs on cultured Ewing's Sarcoma cell lines, we treated Ewing's cells with etoposide, one of the common drugs of the chemotherapeutic cocktail treatment for Ewing's, and with tamoxifen. We used real‐time PCR to analyze mRNA levels of several targets in cells treated with these various chemotherapies. One gene target is GLI1, which is highly expressed in primary tumors. Another target gene sequence is TP53, which is normally a tumor suppressor. mRNA was collected from cells treated with the various chemotherapies andy multiple targets were analyzed. Here we report the results of real time PCR using Taqman probes. Our results contribute to our understanding of how chemotherapies may effect Ewing's Sarcoma tumors and future cultured cell line research in this area.

Support or Funding Information

This work is funded by the College of Saint Scholastica SSC Summer Research Fellowships.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigate Kentucky Hemp‐Induced Modulation of Interleukin‐1 β Secretion in Ovarian Cancer Cells
Authors: Turner Chase , Kenley Colby , Jent Travis , Vu Nam , Hughes Nathan , Sumanasekera Wasana K.
Published: June 2021

Our laboratory is interested in searching for alternative therapies for ovarian cancer. Based on our preliminary studies, KY grown hemp, which is similar to marijuana yet non‐addictive, demonstrated anti‐cancer effects against ovarian cancer. The objective of this study is to investigate the mechanism behind the hemp‐induced anti‐cancer effects. Inflammation is known to play a role in carcinogenesis and Interleukins are contributing players of the inflammation. We hypothesized that the hemp‐induced modulation of Interleukin‐1 β (IL‐1 β) production may play a role in hemp‐induced anti‐cancer effects. Both marijuana and hemp contain therapeutically valuable components such as Cannabidiol (CBD), Cannabidiolic Acid (CBD‐A) and Tetrahydrocannabinol (THC). However, hemp is more beneficial than marijuana due to its' non‐addictive properties. CBD‐A and CBD‐induced modulation of cytokines have been reported in different cell types. To our knowledge, hemp‐induced modulation of ovarian cancer and responsible mechanisms are yet to be discovered.

Methods

KY hemp extracts were made using supercritical CO2 extraction method by KY commonwealth Extracts company. CBD content of KY hemp was analyzed using High Performance Liquid Chromatography (HPLC) by MRX laboratories. In our laboratory, these hemp extracts were tested for its' potential anti‐cancer effects and mechanism of action. A2780 epithelial ovarian cancer cells (OCCs) were seeded in 12–well plates. Following serum starvation, OCCs were treated with either vehicle or different concentrations of hemp extracts for 24 hours. Then, cell supernatants were removed and subject to Bradford protein assay followed by IL‐1 β sandwich ELISA. IL‐1 β released from each treatment group was detected based on manufacturer's protocol. In brief, IL‐1 β standards, cell supernatants from treatment groups, and no hemp negative control were incubated in ELISA plate coated with IL‐1 β antibodies. After a series of steps that included secondary antibodies and a substrate solution, IL‐1 β levels were detected spectrophotometrically. For an experiment, each treatment was done in triplicate and each experiment was repeated a minimum of three times to ensure reproducibility. Hemp‐induced attenuation of IL‐1 β secretion was detected and this attenuation is significant at 500 μg/ml hemp extract treatment, which contains 335 μM CBD and 591 μM CBD‐A. Based on the data, it can be concluded that the hemp extract‐induced attenuation of IL‐1 β secretion might contribute to its protective effects against ovarian cancer.

Support or Funding Information

Funding was provided by Sullivan University System via a faculty development grant awarded to Dr. Sumanasekera. Grant number: RG_1_PS_2017_01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluating the Role of the Na+‐H+ Exchanger Isoform 1 (NHE1) in Lysophosphatidic Acid and Urokinase‐type Plasminogen Activator Stimulation of Ovarian Cancer Cells
Authors: Stiglich Ashley A. , Provost Joseph J. , Wallert Mark
Published: June 2021

Lysophosphatidic Acid (LPA) is a bioactive lipid which stimulates an increase in cell proliferation and migration in a range of normal and cancer lines in part through the activation of both the RhoA/Rock and Ras/ErK signaling pathways. Plasma levels of LPA are elevated in ovarian cancer patients even in the early stages of the disease where it is believed to participate in pathogenesis. In patients with more advanced stages of ovarian cancer, the higher the levels of LPA found in the blood and ascites is a negative prognostic indicator. In ovarian cancer, LPA enhances cell proliferation and promotes cell survival by preventing apoptosis and anoikis. LPA also decreases the sensitivity of the ovarian cancer cells to cisplatin and other chemotherapy drugs. While LPA signaling can directly enhance ovarian cancer progression, it can also act by stimulating the production and release of urokinase‐type plasminogen activator (uPA). uPA is a serine protease that binds to a plasma membrane receptor stimulating activation of both the RhoA/Rock and Ras/ErK signaling pathways. Studies in non‐small cell lung cancer cells have shown that LPA and uPA activate the Na+‐H+ Exchanger Isoform 1 (NHE1) leading to an increase in cell proliferation and migration. NHE1 is an 815 amino acid transmembrane protein that exchanges 1 extracellular Na+ for 1 intracellular H+ leading to the pH inversion common to cancer cells in a developing tumor. Activation of NHE1 is one of the initial steps in the development of the transformed phenotype of cancer cells. NHE1 can be phosphorylated and activated by Rock and kinases in the Ras/Erk signaling pathway. Recently, increases in NHE1 expression was demonstrated to play an important role in cancer progression in ovarian cancer patients. In this project, we specifically explore the relationship between LPA and uPA signaling to NHE1 in the regulation of cell proliferation, stress fiber formation, and cell migration in three ovarian cancer cell lines, CAOV‐3, SKOV‐3, and OVCAR‐3.

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 Role of HSF2 in Human Malignancies
Authors: Takagishi Seesha , Metz Kyle , Wu Yaqi , Alasady Milad , Mendillo Marc
Published: June 2021

Heat‐Shock Factor 1 (HSF1) is the master transcriptional regulator of the heat shock response (HSR), an ancient survival mechanism that is conserved from yeast to humans. In the HSR, proteotoxic stress activates HSF1 to induce expression of heat shock protein genes, including HSP70 and HSP90, to maintain protein homeostasis. In cancer, HSF1 is highly expressed, and drives a transcriptional program distinct from the HSR. Genes in the HSF1 cancer program are involved in a broad range of cellular activities that include protein folding, but also include cell proliferation, cell adhesion, apoptosis, and energy metabolism. To better understand HSF1 function in cancer cells, we used a multifaceted strategy to identify HSF1‐interacting proteins. We found that Heat‐Shock Factor 2 (HSF2), an HSF1 paralog, was among the strongest HSF1 interacting proteins. However, the role of HSF2 in cancer cells and its influence on HSF1 activity is not well understood. We performed RNA‐Seq and ChIP‐Seq to identify the HSF2 cancer program. We used CRISPR‐Cas9 mediated genome editing to knock out HSF2 and HSF1 in a diverse panel of common human cancer cell lines. We found that HSF2 and HSF1 exhibit distinctive effects on transcription, despite sharing a high degree of genomic co‐occupancy. These transcriptional effects were affected by the type of cancer cell line. Lastly, we characterized the HSF2 cancer phenotype in vitro and in vivo. In vitro, we found that the loss of HSF2 affects cell migration and invasion. In vivo, loss of HSF2 affects tumor formation and growth. Our findings suggest a complex interplay between HSF1 and HSF2 that ultimately impacts malignant progression.

Support or Funding Information

NIH/NCI R00 CA175293‐03, Kimmel Scholars Program SKF‐16‐135, Lynn Sage Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endothelial‐derived Extracellular Vesicles Induce Proliferation in Glioblastoma Cells
Authors: Zimmerman Zachary R. , Dores Michael Robert
Published: June 2021

Extracellular vesicles (ECVs) are critical biomarkers for disease and mediate intracellular signaling within the tumor microenvironment. ECVs from tumors enhance cell proliferation and migration in endothelial cells. However, the role of endothelial ECVs in regulating tumor cell proliferation is not well understood. We have investigated the role ECVs play in the communication between glioblastoma cancers and the human endothelium. To determine the effect of ECVs on the communication between glioblastoma multiforme cancer and endothelial cells, ECVs from both cell types were isolated. These ECVs were determined to carry the proteins CD63 and ALIX, known exosome biomarkers and proteins found in extracellular vesicles originating from the multivesicular body. Glioblastoma cells treated with ALIX‐positive exosomes collected from endothelial cells demonstrate increased migration over untreated cells. Signaling through G protein‐coupled receptors modulates ECV content in endothelial exosomes and increases the amount of ALIX‐positive ECVs released. We are investigating whether changing ECV content through GPCR signaling increases their ability to induce proliferation in glioblastoma cells.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Learned Modulation of Innate Odor‐Driven Behavior Requires the Orbitofrontal Cortex
Authors: Miyamoto Kiana , Victoriano Joelle , Kathrotia Mayuri , Root Cory M.
Published: June 2021

Animals use a combination of innate and learned behaviors to survive. Innate behaviors do not require experience and drive stereotypical, hard‐wired responses to particular environmental cues, such as predator odor. However, learning provides a mechanism to integrate past experience and even modulate innate behaviors. We have created a behavioral paradigm to study the learned override of mouse aversion to the fox odor, TMT. The ability of the brain to suppress innate aversion in favor of a learned attraction is a form of executive control. What are the neural circuits involved in this behavior? Our previous work demonstrated that a third order olfactory brain region, the cortical amygdala, mediates innate responses to odor. We hypothesized that a higher order structure should be required to suppress innate aversion and reinforce a positive value. A likely candidate is the orbitalfrontal cortex (OFC), which receives olfactory input from piriform cortex and has outputs to the innate pathways and reward circuitry. The OFC is implicated in executive functions such as value reassignment. We have tested the role of the OFC in modulation of behavioral response to fox odor.

We designed a two‐port decision making task to measure the innate response of mice to attractive or aversive odor stimuli. When odor is paired with an equal water reward at each port, water restricted mice prefer the port with the attractive rose odor, 2‐phenylethanol, to the TMT port. However decreasing the amount of water available at the rose port teaches mice to prefer the fox odor port. The attraction persists even in the absence of water. Thus, mice can learn to be attracted to predator odor that predicts reward.

To test if the OFC is involved in this preference reassignment, we performed a loss of function experiment using optogenetic tools to suppress activity in the neurons of the OFC of trained animals. Bilateral expression of the light gated chloride pump, halorhodopsin, and optical stimulation in the OFC during an unrewarded test phase were sufficient to block the learned behavior and revert the animals to their innate behavioral response of avoiding aversive odor. These findings reveal that the orbitofrontal cortex is indeed required for the learned override of innate behavior. We next plan to record neural activity in the OFC as mice perform the learning task to reveal changes that occur in OFC to accommodate learning, and to map out the relevant downstream targets of OFC.

Support or Funding Information

Grant #: 4R00DC014516‐03 from NIH National Institute on Deafness and Other Communication Disorders (NIDCD)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RGS4 Regulates Neurite Outgrowth And Cell Proliferation Mediated by STAT5B Transcriptional Responses
Authors: Pallaki Paschalina , Serafimidis Ioannis , Papadimitriou Elsa , Papakonstantinou Maria Pagona , Thomaidou Dimitra , Gaitanou Maria , Georgoussi Zafiroula
Published: June 2021

Regulators of G proteins signaling (RGS) serve several cellular functions, despite their initial role as GTPase activating proteins. Recent evidence suggests that RGS proteins interact with transcription factors to mediate transcriptional responses. RGS4, a member of the B/R4 family of RGS proteins, is a multitask protein highly expressed in developing neurons, that has been shown to be a key regulator of opioid receptor signaling (ORs) by confering selectivity for G protein coupling, inhibiting OR‐mediated ERK1,2 phosphorylation and accelerating receptor internalization (Leontiadis et al. 2009; Georgoussi et al. 2006). We have previously shown that δ‐OR forms a multiprotein signaling complex, consisting of Gi/Go proteins and the Signal Transducer and Activator of Transcription 5B (STAT5B) that leads to neurite outgrowth upon DSLET‐δ‐OR activation (Georganta et al. 2010;2013). Based on these findings and the fact that RGS4 directly interacts with STAT5B to negatively regulate STAT5B activation (Pallaki et al. 2017) we wondered whether RGS4‐STAT5B interplay could be involved in cellular mechanisms controlling neurogenesis.

Primary neuronal cortical cultures from RGS4−/− mice, lacking a functional RGS4, exhibited differential neuronal sprouting compared to wild types, as assessed by an increased axonal length and branch number, upon DSLET administration. Additional studies in brain extracts from adult RGS4−/− mice demonstrated increased levels of phospho‐STAT5B while exhibited altered expression levels of STAT5B inducible genes Snx9 and Ptk2 which are both highly implicated in axonogenesis. This result indicates that RGS4 is implicated in neurite outgrowth via a STAT5B‐mediated transcriptional regulation. On the other hand, Neuro2A cells stably expressing RGS4 exhibited a significant reduction in the cell number and proliferation rate whereas isolated RGS4−/− neural stem cells exhibited elevated proliferative properties with a concomitant increase of the mRNA levels of the STAT5B anti‐apoptotic target genes, Bcl‐2 and Bcl‐xl. The same expression pattern was also confirmed in RGS4−/− mature adult brain tissues. Collectively, these studies demonstrate for the first time a novel selective role of RGS4 in STAT5B mediated‐transcriptional responses providing insights into the role of RGS4 involvement in neuronal development and synaptic signaling upon δ‐opioid administration.

Support or Funding Information

This work was supported by grants NORMOLIFE (LSHC‐CT037733) and the GSRT Excellence II, 3722 ‐”NO‐ALGOS” to Z.G. The facilities of the Hellenic Research Infrastructure OPENSCREEN‐GR were used for protein expression.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Developing an Intranasal Colonization Model for NTHi in Mice
Authors: O'Neil Meghan , LaClair Ciara , Zavorin Mark , Pryharski Karin , Khan Nadeem , Kaur Ravinder , Pichichero Michael , Michel Lea Vacca
Published: June 2021

Nontypeable Haeomphilius influenzae (NTHi) is a Gram‐negative pathogenic bacterium that can live as a commensal in the nasopharynx or can be the cause of Acute Otitis Media (AOM). Although there are currently no approved vaccines in the US to protect against NTHi, several NTHi recombinant protein vaccines are currently being studied for their safety and efficacy of protection. To assess the effectiveness of these prototype vaccines, we had to first establish NTHi colonization and AOM infection models in mice. To establish colonization, C57BL/6J mice were intranasally (IN) infected with PR8 (a mouse‐adapted H1N1 virus) prior to IN infecting with NTHi. Several coinfection protocols were tested to optimize NTHi colonization. Bacterial densities were determined at days 3 and 7 post‐NTHi infection by harvesting blood, nasal washes, and ear washes from the infected mice. Using this coinfection strategy, we were successful in developing a robust NTHi nasal colonization model in mice.

Support or Funding Information

Rochester Institute of Technology Honors Program Summer Fellowship and Rochester General Hospital Research Institute Summer Scholars 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.
A comparison of a Natural and Synthetic Stilbenoid, Arachadin‐3, on a Rotavirus Infected Human Intestinal Cell Line
Authors: Napier‐Jameson Rebekah , Witcher Caleb M. , Wisdom Stormey B. , Strange Essence B. , Triggs Doran S. , Saade Luanna L. , Taylor Josephine , Ball Judith M. , Medina‐Bolivar Fabrecio , Parr Rebecca D.
Published: June 2021

Rotavirus (RV) causes severe diarrhea in young children and immunocompromised individuals. Several effective vaccines have been developed to immunize children between 2 to 8 months of age, but there are no vaccines or antiviral therapeutics for RV‐infected immunocompromised patients of any age. Our laboratory has shown a decrease in the number of infectious RV particles produced in HT29.f8 cells with the addition of the stilbenoid, arachidin‐3 (A3). Additionally, the viral protein, NSP4, that is important for viral replication is decreased, suggesting an effect on virus replication. Also, ultrastructural changes were observed with TEM studies that imply a connection with the apoptosis and autophagy pathways which is supported by qRT PCR data. Recently, a synthetic A3 (sA3) has been developed. This study compared the effects of the natural A3 and sA3 on HT29.f8 cell viability, production of infectious RV particles, production of structural and nonstructural viral proteins, ultrastructural changes and the gene regulation of apoptosis and autophagy transcripts. From the data collected, the viability of the cells was not affected with the introduction of either natural or sA3, but the amount of infectious virus particles produced was significantly decreased by 50‐fold, respectively. The TEM images depicted changes in ultrastructures of RV‐infected HT29.f8 cells that appear to show signs of apoptosis while RV‐infected cells with the addition of either the natural or synthetic A3 showed a more normal appearance like the negative controls. The nucleus to cytoplasm ratios of TEM micrographs of RV‐infected cells was greater than 1.0 whereas the arachidin treated RV‐infected cells was about 0.5 which was like the control cells. To validate this data, whole cell staining of the nucleus and cytoplasm showed similar changes with the addition of both the A3 and sA3. qRT PCR analysis of transcripts for genes that are important in regulating the apoptosis and autophagy pathways regulations corroborated the TEM observation, and suggests a cross talk between the two pathways during an RV infection that is regulated with the addition of the arachidins. Our data suggests that both the natural A3 and sA3 have anti‐RV activity that implies a potential therapeutic application.

Support or Funding Information

This work was supported by: 1) The Faculty‐Student Collaborative Research Program of The Office of Research and Sponsored Programs at Stephen F. Austin State University and 2) The NSF‐EPSCoR (grant# EPS‐ 0701890; Center for Plant‐Powered Production‐P3).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RASG12D causes more Proliferation than RASG12V in Drosophila Pancreatic Cancer Models
Authors: Bernard Faviola , Sonoshita Masahiro , Cagan Ross
Published: June 2021

Pancreatic cancer is the fourth leading cause of death in the United States. Over 95% of pancreatic cancer patients have activating mutations in the KRAS oncogene, suggesting that these mutant KRAS forms play a critical role in development of the disease. The most common mutations in KRAS are G12D and G12V missense mutations, but little is known about functional differences between them. Therefore, our objective is to compare the outcomes between KRASG12D and KRASG12V mutations in pancreatic cancer models. Using the Gal4‐UAS system, which allows the expression of transgenes in a specific tissue, we developed pancreatic cancer models in the fruit fly Drosophila. The patched promoter‐driven Gal4 transcription factor targeted the Drosophila RasG12D or RasG12V transgene to the midline of the fly wing epithelium. Using green fluorescence protein (GFP) as a marker for Ras‐expressing cells, we found that RASG12D directed more aggressive expansion of the patched domain than wild type or RASG12V wing epithelia. Oral dosing of trametinib—a MEK inhibitor that blocks signaling downstream of RAS—decreased proliferation of RASG12D expressing cells. These results indicate that trametinib could be a useful drug against RAS‐positive cells for RASG12D mutations. Future experiments include understanding the mechanisms by which RASG12D and RASG12V direct different phenotypes, and further developing novel treatments based on trametinib.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antimycobacterial and macrophage apoptosis inducing effects of Psychotria capensis and Psychotria zombamonatana species
Authors: Aro Abimbola , Fonteh Pascaline , McGaw Lyndy Joy
Published: June 2021

The causative agent of tuberculosis (TB), Mycobacterium tuberculosis (Mtb), has succeeded in infecting one‐third of the human race through inhibition or evasion of the innate and adaptive immunity. Mtb was responsible for 1.5 million deaths and the emergence of 9.6 million new cases of TB in 2014. Apoptosis is a cellular biology process that is closely involved in the interaction between Mtb and the phagocytes it infects such as macrophages, dendritic cells and neutrophils. It has been suggested that apoptosis of infected macrophages is one way in which the host deals with intracellular pathogens. To effectively combat these disease, new TB drugs with novel modes of action different from the presently administered drugs are urgently needed. Approximately 65% of antibacterial approved for use between 1981 and 2010 were natural products or natural product derivatives including currently employed TB drugs such as rifampicin and the aminoglycosides The acetone extracts of Psychotria capensis and Psychotria zombamontana have been reported to be active against non‐pathogenic mycobacteria This study aims to assess the antimycobacterial activity of P. capensis and P. zombamontana against pathogenic strains of mycobacteria and also to shed some light on elucidating the anti‐tuberculosis mechanism of the selected plant extracts.

The antimycobacterial activities of the crude extracts of these two species belonging to the rubiaceae family were screened against Mycobacterium tuberculosis ATCC, Mycobacterium bovis ATCC and H37Rv strains to determine the minimum inhibitory concentration (MIC). THP‐1 cells were treated with varying concentrations (20, 50, 100 μg/mL) of each extract for 24 and 48 h. Cell apoptosis was measured by annexin V/propidium iodide apoptosis kits using flow cytometry. The concentration of IL‐12 in the supernatant of the cell culture was determined by using the human enzyme linked immunosorbent assay (ELISA) to assess the immune modulatory effect of the extracts.

The acetone crude extracts of the plant species had an excellent antimycobactrial activity against the tested mycobacteria strains with a MIC values ranging from 78–156 μg/mL. P. capensis and P. zombamontana extracts induced apoptotic effects on THP‐1 cells following treatment in a time and dose‐dependent manner. Interestingly, the acetone extracts of P. capensis revealed a profound macrophage apoptosis induction (16%) and also enhanced the expression of IL‐12 in a dose dependent manner. Results obtained from this study suggest that the acetone extracts of P. capensis may contain promising lead compounds for anti‐TB therapy. There is on‐going work to isolate compounds and analogues responsible for the observed anti‐TB activity.

Support or Funding Information

University of Pretoria, South Africa

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