bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of HDAC inhibitor Vorinostat and radiation on HTB4 and MDA‐MB‐435 cancer cells
Authors: Wing Joseph Francis , Maini Trisha , O'Donnell Robert Watts
Published: June 2021

HDAC inhibitors have shown promise in the treatment of various types of cancer. The HDAC inhibitor vorinostat was first introduced during treatments for cutaneous T‐cell lymphoma and has been shown to cause an increase in the effectiveness of radiation on multiple cancer cell lines. For the purpose of these experiments, the effects of vorinostat and radiation on HTB4 cells (a human bladder cancer cell line) and MDA‐MB‐435 cells (a human melanoma cell line) are being investigated in order to determine the efficacy of this combined treatment. Using cell cycle analysis and MTT assays, we were able to analyze the effects of radiation and vorinostat alone compared to the combination of both. For cell cycle analysis, we plated the cells in four flasks and treated three of them with either vorinostat (2.5 μM) alone, radiation (~20Gy) alone or the combination of the two. The cells were harvested, stained and analyzed five hours after radiation and 24 hours after vorinostat treatment by flow cytometry. Preliminary results for the HTB4 cells treated with both radiation and vorinostat showed an increase in the percent of cells in G2 and a reduction in the percent of those in both G1 and S phase compared to either treatment alone or the control population. Results for the 435's did not show a similar trend with distribution of cells between the control and three treatments. The MTT assay was performed using two 96‐well microtiter plates with 16μM vorinostat serially diluted 1:2 across 10 columns, and only cells in the 11th column and medium alone in the 12th column. One plate was irradiated using a lower dose of 5 Gy based on a literature survey. MTT was added to both plates directly after the one plate was irradiated. Results of the MTT assay for HTB4's and 435's showed that increasing concentrations of vorinostat alone and when coupled with radiation resulted in less MTT product produced compared to the radiation treatment alone. Based on the MTT data, the HTB4 cell line appears to be more sensitive than the 435's to the effects of vorinostat. Future experiments will focus on the effects of radiation on multiple tumor cell lines and investigate the molecular mechanisms by which HDAC inhibition influences radiation‐based cancer treatment.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using Bacterial Transcriptomics to Investigate Targets of Host‐Bacterial Interactions in C. elegans
Authors: Wong Hoi Tong , Chan Jason P. , Brumbaugh Jamey T. , Ardasheva Anastasia , McLimans Christopher J. , Wright Justin , Lamendella Regina
Published: June 2021

The interactions between a host and its resident microbes form complicated networks and disentangling these interactions can help us better understand mechanisms of pathogenesis while also defining the integral commensal protection that host‐associated microbiota offer to promote health. Here we utilize a tractable model organism, C. elegans, to study the mechanisms involved in host‐microbe interactions and to identify different expression profiles under different host genotypes to increase our knowledge of host‐bacteria interspecies interactions. We developed an RNAseq protocol and bioinformatics pipeline to elucidate 1) bacterial gene expression differences in host associated versus in vitro E. coli OP50 and 2) differential bacterial gene expression in known models of aging within C. elegans (long‐lived daf‐2/InsR mutant versus short‐lived daf‐16/FOXO transcription factor mutant). Briefly, RNA extracts (n=18) were subject to ribosomal RNA subtraction, cDNA synthesis, Nextera library preparation and sequencing using the Illumina Hiseq platform. Our bioinformatics pipeline utilizes the most robust programs for quality filtering, assembly, annotation, and differential gene analysis. Our analysis revealed a variety of differentially expressed genes in plated E. coli OP50 versus E. coli OP50 fed to C. elegans. Differential gene expression analysis revealed that expression of biosynthetic genes was enriched in host‐associated E. coli, and several of these expressed genes code for the precursors and products needed for the synthesis of Lipopolysaccharides (LPS), which are important in innate immune and stress responses, as well as pathogenicity. Preliminary bacterial transcriptome data also indicate differences in bacterial expression of several biosynthetic pathways and virulence mechanisms, which may contribute to the aging phenotypes of the long‐lived daf‐2/InsR mutant and the short‐lived daf‐16/FOXO transcription factor mutant. Our research has employed a robust RNA sequencing and bioinformatics approach to study differential gene expression of bacteria in C. elegans, and will enable us to better test how gut bacteria and their gene regulatory networks regulate host physiology.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Caloric restriction impacts plasma microRNAs in rhesus monkeys
Authors: Clark Josef , Schneider Augusto , Dhahbi Joseph , Atamna Hani , Colman Ricki , Anderson Rozalyn
Published: June 2021

Caloric restriction (CR) is one of the most robust interventions shown to delay aging in diverse species, including rhesus monkeys (Macaca mulatta). Although the mechanisms have yet to be elucidated, identification of factors involved in delayed aging by CR brings a promise of translatability to human health and human aging. Here we show that CR in rhesus monkeys induced a profound change in abundance of circulating microRNAs (miRNAs) linked to growth and insulin signaling pathway, suggesting that miRNAs are involved in CR's mechanisms of action in primates. Deep sequencing of plasma RNA extracts enriched for short species revealed a total of 243 unique species of miRNAs including 47 novel species. Approximately 70% of the plasma miRNAs detected were conserved between rhesus monkeys and humans. CR induced or repressed 24 known and 10 novel miRNA species. Regression analysis revealed correlations between bodyweight, adiposity, and insulin sensitivity for 10 of the CR‐regulated known miRNAs. Sequence alignment and target identification for these 10 miRNAs identifies a role in signaling downstream of the insulin receptor. The highly abundant miR‐125a‐5p correlated positively with adiposity and negatively with insulin sensitivity, and was negatively regulated by CR. Putative target pathways of CR‐associated miRNAs were highly enriched for growth and insulin signaling that have been previously been implicated in delayed aging. Clustering analysis further pointed to CR‐induced miRNA regulation of ribosomal, mitochondrial, and spliceosomal pathways. These data are consistent with a model where CR recruits miRNA‐based homeostatic mechanisms to coordinate a program of delayed aging.

Support or Funding Information

NIH/NIA AG040178, AG047358, and the Glenn Foundation for Medical Research. JPC is a T32 Fellow NIH/NIA AG000213

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interactions between Divalent Cations and a Periplasmic Lipoprotein involved in Salmonella Magnesium Homeostasis
Authors: Soens Ross , May John
Published: June 2021

All living cells require magnesium to generate biochemical energy during metabolism and for maintenance of essential structures. A recently discovered protein of unknown function, DcrB, plays a role in magnesium homeostasis within Salmonella enterica, which must adapt to low external levels of Mg2+ to cause food‐borne illness. DcrB is a small protein attached to the periplasmic face of the cytoplasmic membrane. Given this location, one possible model for the function of DcrB is that DcrB brings Mg2+ to membrane Mg2+ transporters in the cytoplasmic membrane. Currently, Salmonella phenotypic assays have shown DcrB to promote growth in low Mg2+. The hypothesis of this project is that DcrB binds magnesium and possibly other divalent cations. To test this hypothesis, we examined whether Mg2+ and other divalent cations affected the thermal stability of purified DcrB protein. We examined the effect of divalent cations using a fluorescence‐based thermal shift assay and circular dichroism techniques. Furthermore, we are using our recent crystallographic structure of DcrB to identify and test specific metal binding sites. Our results have established the specificity of DcrB for binding divalent cations, which we anticipate will shed light on the role of DcrB in metal homeostasis in pathogenic bacteria.

Support or Funding Information

This research is supported by a University of Wisconsin‐‐La Crosse Undergraduate Research and Creativity Grant (RS) and a University of Wisconsin‐‐La Crosse Faculty Research Grant (JM).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Analysis of Cortactin After Anion Exchange Chromatography
Authors: Graves Gabriel , Kruchten Anne
Published: June 2021

Cortactin is a cytoskeletal actin‐binding protein that is upregulated in several types of metastatic cancers. Further research on its three‐dimensional structure may allow for greater understanding of its role in cancers. Purification of our in vitro expressed His‐tagged cortactin from E. coli was done through affinity purification with Ni2+ resin followed by anion exchange chromatography. Elution fractions of the protein were analyzed via polyacrylamide gels both in native and SDS environments to analyze both length and hydrodynamic size of the protein. Silver staining of subsequent gels allowed for precise analysis of banding pattern and relative strength. Our results demonstrate variations in banding patterns with increasing concentration of KCl elution on native‐PAGE, suggesting cortactin has a variable three dimensional structure in vitro. Future work will determine if these structural differences are maintained in vivo.

Support or Funding Information

This work is supported 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.
Studying protein‐protein interactions in fatty acid and polyketide biosynthetic pathways via site‐specific vibrational spectroscopy
Authors: Chioti Vasiliki T. , Londergan Casey H. , Charkoudian Louise K.
Published: June 2021

By virtue of their structural diversity and striking potency, natural products have long been used as therapeutic agents. Natural products are biosynthesized by assemblies of enzymes encoded in gene clusters. Repurposing the natural product biosynthetic repertoire to assemble “unnatural” natural products would have profound implications for human health. Our current lack of understanding of how these biosynthetic enzymes operate at a molecular level, which is partially attributed to the lack of tools that can capture the weak and transient interactions of the enzyme assemblies, stymies such engineering endeavors. Our lab previously showed that labeling acyl carrier proteins, which are central hubs of natural product assemblies, with a thiocyanate (SCN) vibrational probe provides an innovative method to study the proteins' conformational dynamics and interactions with ketosynthases. In this work, we evaluate the general utility of the probe by assessing ACP‐SCN interactions with other partner enzymes. The SCN probe was loaded onto the 4′‐phosphopantetheine (Ppant) arm of ACPs involved in fatty acid and actinorhodin type II polyketide biosynthetic pathways, AcpP and actACP, respectively. Changes in the CN stretching frequency were monitored as the ACPs were titrated with partner enzymes, 3‐hydroxyacyl‐ACP dehydrase (FabA) and actinorhodin polyketide ketoreductase (actKR). Upon a productive interaction of the ACP with its respective partner enzyme, the CN absorption band shifts to lower wavenumbers, which could correlate to the probe accessing the partner enzyme's hydrophobic catalytic active site. In light of these findings, the SCN probe appears to report on a wide range of ACP‐partner protein interactions. As a result, the probe could be a useful tool for future engineering studies.

Support or Funding Information

We would like to acknowledge an NIH Award #CHE‐1652424 to Louise K. Charkoudian as a source of funding for this project.

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 role for the N‐terminal domain in modulating the activities of the nucleotide exchange factor Sil1
Authors: Pareja Kristeen Alcaide , Sevier Carolyn
Published: June 2021

The Hsp70 BiP is an essential molecular chaperone in the endoplasmic reticulum (ER) and is highly involved in maintaining the quality of proteins in the secretory pathway. Using yeast as our model system, we have previously shown that BiP is also involved in ER oxidative stress signaling. BiP contains a conserved cysteine located in its nucleotide binding domain, and this cysteine can be modified by peroxide (sulfenylated) and/or glutathione (glutathionylated) when the ER experiences an overly‐oxidizing environment. Normally, BiP undergoes ATP‐dependent conformational changes that drive the association and dissociation of BiP with polypeptides. We have shown that the cysteine modification limits BiP's ATP hydrolysis yet BiP maintains its ability to bind substrates. We have demonstrated that this ATP‐independent holdase activity of modified BiP is beneficial to cells under conditions of ER oxidative stress, likely a consequence of limiting polypeptide aggregation under suboptimal folding conditions. When cells go back to non‐stress conditions, reversibility of BiP's cysteine modification is also important for normal cellular function and survival. The protein Sil1, a known nucleotide exchange factor (NEF) of BiP, can remove the modification on BiP's cysteine. Sil1's unexpected activity as a reductant depends on two cysteines within its N‐terminal domain: Cys‐52 and Cys‐57. Although the N‐terminal domain containing the cysteines is required for Sil1 activity as a reductant, Sil1 is competent as a NEF in the absence of the N‐terminal domain. Mutations in the Sil1 gene are major cause of Marinesco‐Sjögren Syndrome (MSS), a rare autosomal disease characterized by early onset cataracts, cerebellar ataxia, and delayed mental development. Interestingly, we found a patient mutation that maps within the N‐terminal region which not only influences Sil1's reductant activity but also dramatically alters Sil1's activity as a NEF. We are in the process of further elucidating the molecular details relating to how the N‐terminal region of Sil1 impacts both its NEF and reductant activity by utilizing a variety of biochemical and biophysical approaches. Our current data suggest that the N‐terminal domain adopts multiple conformations, allowing for regulation of Sil1's NEF activity and potentially its activity as a reductant.

Support or Funding Information

NSF GRFP, SUNY Diversity Fellowship, NIGMS

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Complex Effects of J‐protein Alterations on Hsp104‐mediated Curing of Prion [PSI+]
Authors: Berger Scott E. , Kamiya Erina , Astor Michael T. , Hines Justin K.
Published: June 2021

The amyloid‐based prions of Saccharomyces cerevisiae are heritable aggregates of misfolded protein, passed to daughter cells following fragmentation by a set of molecular chaperones which includes the J‐protein Sis1, Hsp70, and Hsp104. Overexpression of Hsp104 efficiently cures the prion [PSI+], a phenomenon which has promoted the exploration of Hsp104 as a potential therapeutic agent for neurodegenerative diseases. However, the mechanism of [PSI+] elimination by Hsp104 overexpression has been the subject of significant debate for the past two decades and has garnered significant interest in the recent literature as multiple conflicting models have been proposed. Yeast prion propagation is inexorably reliant on the function of molecular chaperones of the Hsp100, Hsp70, and Hsp40 classes. Specifically, four Hsp40s (also called J‐proteins) have been implicated in various aspects of yeast prion biology: Sis1, Ydj1, Apj1, and Swa2. We found that overexpression of Sis1 or Apj1 accelerates strong [PSI+] elimination by Hsp104 overexpression, yet Ydj1 overexpression has a profound and opposing effect, completely blocking Hsp104‐mediated curing, indicating that Apj1 and Sis1 likely have similar and partially overlapping roles in this process. Interestingly results for weak variants of [PSI+] indicated potentially no role for J‐proteins in curing as no J‐protein alteration whatsoever affected the ability of Hsp104 to cure these variants. Additional experiments to determine the specific J‐protein domains responsible for various effects, as well as J‐protein requirements in cell backgrounds harboring both [PSI+] and [RNQ+] are underway. Overall our data support the hypothesis that Hsp104‐mediated curing may occur by biochemically distinct, variant‐specific mechanisms, only some of which involve J‐proteins.

Support or Funding Information

This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM110606. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increasing Surface Expression of Pancreatic β cell‐KATP Channels Attenuates Palmitic acid‐induced Lipotoxicity in vivo and in vitro
Authors: Chen Pei‐Chun , Ruan Jing‐Syuna , Kuo Yi‐Ying , Chao Ru‐Yi , Chen Yun‐Wen
Published: June 2021

In pancreatic β‐cells, KATP channels serve as glucose sensors and effectively initiate glucose stimulated insulin secretion (GSIS). When glucose enters β‐cell via glucose transporter 2, glucose metabolism increases intracellular ratio of ATP to ADP and in turn closes KATP channels which are composed of 4 sulfonylurea receptor 1 (SUR1) and 4 inwardly rectifying potassium channel 6.2 (Kir6.2) subunits. Chronic PA‐injected mice displayed type II diabetic characteristics including hyperglycemia and hyperinsulinemia. The pancreatic sections of these mice upregulated expression of cleaved caspase 3 and calnexin and importantly reduced surface expression of SUR1. Then, we tested time and dose effects of PA on the cell viability using INS‐1 cells. We observed the significant decrease in the surface expression of SUR1 after 72 hours of 0.4 mM PA. In addition, this treatment induced pancreatic b cells apoptosis by increasing cleaved caspase 3 proteins. Our results showed GB increased surface expression of SUR1 and decreased pancreatic β cell death and recovered insulin secretion upon glucose stimulation. Co‐treatment with GB in PA‐treated cells reduced ER stress and recovered mitochondrial membrane potential. Importantly, GB also improved glucose tolerance, insulin resistance, decreased triglyceride concentration and increased insulin release in PA‐injected mice. In conclusion, correct trafficking of KATP channels improved pancreatic β‐cell function and increased insulin sensitivity.

Support or Funding Information

We thank Dr. Christopher Newgard for the INS‐1 cell clone 832/13. We also thank the staffs of the Advanced Light Microscopy Core at National Cheng Kung University Hospital and Wallace Academic Editing for their help with this manuscript. This research received financial support from the Ministry of Science and Technology (MOST103‐2320‐B‐006‐005‐MY2, MOST105‐2628‐B‐006‐006‐MY3, MOST106‐2320‐B‐006‐050) offered to Pei‐Chun Chen.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of several PD‐associated genes on the toxicity of a‐synuclein in a yeast model
Authors: Jones Paul Andrew , Ong Emily , Balaram Ariane , Biel Alexsandra , Tembo Maiwase , DebBurman Shubhik
Published: June 2021

Parkinson's disease (PD) is characterized by a‐synuclein misfolding and the death of midbrain neurons. PD can be described as familial, or sporadic, both of which are influenced by a multitude of environmental and genetic factors. Familial PD is directly caused by a mutation in one of at least ten genes, including SNCA, DJ‐1, VPS35, and ATP13A2. SNCA, which encodes a‐synuclein, has six identified missense mutations (A30P, E46K, H50Q, G51D, A53E, and A53T) that each cause autosomal dominant PD. Sporadic PD is linked with several risk genes and loci, including VPS13, the Sac I domain of SYNJ1, and the Swa2 domain of DNAJC6. Using our previously established budding yeast model system for α‐synuclein, we first show that wild‐type (WT), E46K, A53T, H50Q, and A53E a‐synuclein are toxic to yeast and show varying degrees of membrane binding and aggregation, while A30P and G51D a‐synuclein are relatively non‐toxic and shows cytoplasmic diffuse localization. What is still not well understood is whether the other PD‐causing and risk genes mentioned above can influence toxicity and localization properties of WT a‐synuclein and these six familial PD mutants. To test the hypothesis that they do influence a‐synuclein, WT and familial mutant forms of a‐synuclein were studied in haploid yeast strains that were singly deleted for these six PD‐linked genes (all of which are linked to loss‐of‐function in PD). Results show that some gene deletions increase (Δhsp31) or decrease (Δatp13, Δvps35) a‐synuclein toxicity and alter its localization in a highly familial mutant specific way, while others more broadly increase a‐synuclein toxicity or aggregation (Δvps13, Δsac1), while still others no effect (Δswa2). Our findings suggest that WT and each familial mutant of a‐synuclein create cellular toxicity and alter localization in distinct ways and that each is likely regulated by different subsets of genes, opening doors for mutant‐specific mechanistic insight into the varying modes of a‐synuclein toxicity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of immobilization on the specificity and stability of choline oxidase
Authors: Fischel Adam H. , Holton Kortnie , Schmitz Jonathan M. , Watkins Linette M.
Published: June 2021

Choline oxidase is a versatile enzyme that has uses in both industrial and medicinal settings. Choline oxidase is able to act as a biosensor and detect the presence of choline, as well as being able to produce glycine betaine, which is used in various industrial settings. In order for choline oxidase to be feasible in industrial synthesis pathways, it must have a broad specificity while maintaining activity over a range of pH and temperature. To this end, the specificity for two different substrates, temperature stability, and pH stability of choline oxidase were all determined. Following this, choline oxidase was immobilized through two different methods, by attachment to CnBr‐activated Sepharose beads, and by binding to a Polyethersulfone (PES) membrane. When immobilized with Sepharose beads, the temperature and pH stability of choline oxidase increased, while reducing the enzymes specificity for choline as a substrate at the same time.

Support or Funding Information

This work was supported by the James Madison University Department of Chemistry and Biochemistry and the Huntsman Corporation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of the Substitutes on α‐(N)‐Heterocyclic Thiosemicarbazones that Interact with Human Topoisomerase‐II‐α with Nuclear Magnetic Resonance
Authors: Hill Jessica , Conner Jennifer , Combs Nathan , Bowman Sarah , Carroll William , Lisic Edward , Jiang Xiaohua
Published: June 2021

Topoisomerase IIα (TopoIIα) is an essential enzyme for cell proliferation. Cancer cells are rapidly dividing cells. Thus, TopoIIα is a primary target for many anticancer drugs. Thiosemicarbazones (TSCs) have been known to possess anticancer activity by inhibiting topoisomerase‐II. Recent studies of our lab found the structure‐activity relationship of TSCs and their inhibition on TopoIIα. Our results suggest that the metal ion of TSC metal complexes and the terminal nitrogen side chain play important roles in increasing TopoIIα‐mediated cleavage complexes. The direct interaction between TSCs and TopoIIα remains unknown. It is hypothesized by our lab that the metal ions and the terminal nitrogen side chains interact with TopoIIα directly. This hypothesis is being tested by studying the interaction of 2‐acetylthiazole (ATZ) TSC, 2‐acetyl‐4‐methylthiazole (AMT) TSC, and benzoylpyridine (BZP) TSC metal complexes with TopoIIα by saturation transfer difference nuclear magnetic resonance (STD‐NMR) and diffusion ordered spectroscopy nuclear magnetic resonance (DOSY‐NMR). Recent STD‐NMR experiments suggest that the aliphatic region of 2‐acetylthiazole (ATZ) TSC and benzoylpyridine (BZP) TSC interact with TopoIIα.

Support or Funding Information

This research is supported by the Faculty Research Grant (XJ) and the NSF MRI project (1531870).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of methodology for the analysis of novel compound RpM
Authors: Fraser Andrew , Reddy E Shyam P. , Sarkar Arindam Basu
Published: June 2021

Objective

This study was carried out to develop the analytical methodology and limited characterization of the novel compound codenamed RpM. The structure of the novel molecule is being withheld to protect the intellectual rights and patent issues involved.

Introduction

The molecule under investigation, codenamed RpM, is a novel molecule that is custom manufactured for research purpose only. It is believed to have anti‐cancer activity and thus is a subject of interest and investigation.

Method

The UV‐Visble absorption spectra of the compound is established. A reverse phase liquid chromatographic method is developed to separate RpM from its close structural analogs code named RmBr, RpM and RPh using a Xorbax Eclipse XDB‐C18 as stationary phase. The mobile phase is a combination of acetonitrile and water used in a continuously changing concentration gradient. Standard curve was prepared between concentrations of 0.5–10 mcg/ml with RmBr as internal standard; limit of detection (LOD) and quantitation (LOQ) was established. Statistical analysis of the data was carried out. The solubility of the compound in water, water containing 5% DMSO and water containing 5% DMSO and 5% Cremaphor was established by vortexing and ultra‐centrifugation. The stability profile of the compound in water and water containing 5% DMSO and 5% Cremaphor is established in glass container.

Results

Within the specified range the standard curve yielded a slope of 0.2231 (±0.0039) and an intercept of 0.0014 (±0.0058) and good linearity (R2≥0.999). RpM solubilities are found to be 0.1, 0.27, >5 mcg/ml in water, 5% DMSO and 5% DMSO+5% Cremaphor respectively. The compound is found to be stable (≥90% of original content) for at least 7 and days 5% DMSO and 5% DMSO+5% Cremaphor respectively. The stability is water is not determined because of the limited solubility of the compound in water.

Conclusion

The project is successful in establishing an analytical methodology for RpM that is repeatable, robust, accurate and precise in the range. The solubility and stability of the compound in common aqueous based solvents have been established. More work is required achieve a complete physico‐chemical characterization of compound.

Support or Funding Information

*This work has been financially supported by University of Findlay, College of Pharmacy funds.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Physico‐chemical Characterization of Novel Compound RmBr
Authors: Higley Courtney , Sarkar Arindam Basu , Reddy E Shyam P.
Published: June 2021

Objective

This study was carried out to develop the analytical methodology and limited characterization of the novel compound codenamed RmBr. The structure of the novel molecule is being withheld to protect the intellectual rights and patent issues involved.

Introduction

The molecule under investigation, codenamed RmBr, is a novel molecule that is custom manufactured for research purpose only. It is believed to have anti‐cancer activity and thus is a subject of interest and investigation.

Method

The UV‐Visble absorption spectra of the compound is established. A reverse phase liquid chromatographic method is developed to separate RmBr from its close structural analogs code named RmBr, RmBr and RPh using a Xorbax Eclipse XDB‐C18 as stationary phase. The mobile phase is a combination of acetonitrile and water used in a continuously changing concentration gradient. Standard curve was prepared between concentrations of 0.5–10 mcg/ml with RmBr as internal standard; limit of detection (LOD) and quantitation (LOQ) was established. Statistical analysis of the data was carried out. The solubility of the compound in water, water containing 5% DMSO and water containing 5% DMSO and 5% Cremaphor was established by vortexing and ultra‐centrifugation. The stability profile of the compound in water and water containing 5% DMSO and 5% Cremaphor (a commonly used solubilizing agent) is established in glass container.

Results

Within the specified range the standard curve yielded a slope of 55.62 (±1.57) and an intercept of −0.05 (±1.28) and good linearity (R2≥0.999). RmBr solubilities are found to be 0.055, 0.16 nd more than 5 mcg/ml in water, 5% DMSO and 5% DMSO+5% Cremaphor respectively. The compound is found to be stable (≥90% of original content) for at least 7 and days in both 5% DMSO and 5% DMSO+5% Cremaphor. The stability is water is not determined because of the limited solubility of the compound.

Conclusion

The project is successful in establishing an analytical methodology for RmBr that is repeatable, robust, accurate and precise in the range. The solubility and stability of the compound in common aqueous based solvents have been established. More work is required achieve a complete physico‐chemical characterization of compound.

Support or Funding Information

*This work has been financially supported by University of Findlay, College of Pharmacy funds

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Delivery of Interference RNA Molecules to Ovarian Cancer Cells Using Gold‐Liposome Nanoparticle Conjugates
Authors: Quiñones‐Díaz Blanca I. , Grafals‐Ruiz Nilmary , Barletta‐Bonanno Gabriel L. , Vivas‐Mejía Pablo E.
Published: June 2021

RNA interference is a therapeutic modality in which RNA molecules, such as small interference RNAs (siRNAs) and microRNAs (miRNAs) (inhibitors and mimics), are used to regulate the expression of target mRNAs. MiRNAs are multitargeting RNA molecules that are involved in the regulation of several targets and can therefore influence multiple pathways when deregulated. On the other hand, siRNAs repress a specific gene by sequence complementarity to the target mRNA. Although both miRNAs and siRNAs have an increasing therapeutic potential, systemic delivery of RNA‐based technologies is limited by the poor stability of RNA molecules in circulation. Hence, our main goal is to develop a carrier for the efficient and targeted delivery of RNA molecules to ovarian cancer cells. To achieve this, we propose a nanocarrier formulation of gold nanoparticle (AuNP)‐RNA conjugates encapsulated on folate receptor targeted liposomes that could achieve a double protection layer for the RNA while increasing delivery to ovarian cancer cells that overexpress the folate receptor on their surface. We prepared carriers for siRNAs and miRNA oligonucleotide mimics by conjugation of the RNA molecules to the surface of 15 nm diameter AuNPs via thiol linkages. We performed this conjugation in presence of polyethylene glycol (PEG‐2000) to avoid gold precipitation. Physical characterization of the carriers was conducted by dynamic light scattering (DLS) and quantification of conjugated RNA was assessed by fluorescence. The conjugation resulted in approximately 47 to 75 RNA molecules linked per nanoparticle and conjugates of 20 to 25 nm diameter with a slightly negative charge. In addition, encapsulation of the AuNP‐RNA conjugates into liposomes was attained with more than 70% encapsulation efficiency. These results indicate the feasibility of the development and production of our nanocarrier. As a proof of principle we are using this nanoparticle conjugate to target Integrin‐linked kinase (ILK). ILK is a pseudokinase that has been found to be aberrantly expressed in ovarian tumors. Further experiments of internalization, stability and toxicity demonstrate the efficacy of this nanocarrier as a delivery system for miRNA mimics and siRNAs to ovarian cancer cells. We conclude that siRNA‐based technologies are an alternative strategy to target ILK and other target proteins lacking specific chemical inhibitors.

Support or Funding Information

RCMI: U54 MD007600

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Validation of In Vivo Protein Surface Accessibility Method
Authors: Ma Michelle , Bamberger Casimir , Martínez‐Bartolomé Salvador , Yates John R.
Published: June 2021

Measuring changes in surface accessibility is a way to probe conformational changes of proteins. Traditionally, in vitro methods of resolving protein structures have relied on the isolation and purification of proteins, but this artificial protein environment may not reflect its functional native conformation. Therefore, this project seeks to develop a novel approach to identify and quantify changes in protein surface accessibility caused by protein misfolding or altered biomolecule interactions in vivo on a proteome‐wide scale. We want to find out if this approach, chemically labeling the ɛ‐amino group of lysine residues, will yield the same results in vitro as it did in vivo, thus validating the accuracy of results generated with this technique. This dimethylation method is expected to yield biologically relevant information as it allows for the screening of proteomes for structural perturbations as well as identification of novel structural information for proteins not yet crystallized.

Using GAPDH as a control, we showed that the GAPDH tetramer stays intact upon dimethylation using native gel electrophoresis. Using data from crystal structures of GAPDH, the GAPDH tetramer contains one predicted inaccessible lysine residue and we were able to detect this inaccessible lysine residue with our in vitro dimethylation experiment. In addition, under time‐limited conditions, the dimethylation reaction does not go to completion and appears to preferentially occur at certain lysine sites. The extent of dimethylation does not scale with relative surface accessibility and rather requires a minimal accessibility to the ɛ‐amino group of lysine residues.

The dimethylation labeling method is reproducible in vitro as well as in vivo. The GAPDH experiments show that the method chemically labels proteins that are still in their native conformation. Limiting conditions of the dimethylation reaction provides additional structural information of the protein, which allow us to validate predicted structural models and/or data. This technique can be utilized to screen the proteome for structural alterations, which is especially important in diseases or drug interactions.

Support or Funding Information

NIH NCI Enabling Technologies Grant (IMAT) R33CA212973‐01 to John R. Yates III

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antiproliferative Effects of Soy Derived Dipeptides in Human Breast Cancer Cell Lines
Authors: Das Salil K. , Mitra Piyali , Maebuchi Motohiro , Mukherjee Shyamali
Published: June 2021

In the United States, breast cancer is the second leading cause of cancer‐related deaths in women. Diet is an important factor in the development of numerous diseases, including breast cancer. We reported earlier that the consumption of soy protein controlled not only the proliferation of dimethylbenz [a] anthracene (DMBA)‐induced breast cancer in rats but also its aggressiveness. Soy protein is hydrolyzed in the gut to small peptides which are easily absorbed and are involved in various cellular functions. Fuji Oil Company, Osaka, Japan, has commercially marketed a soy protein hydrolysate, termed Hinute‐AM, composed of primarily di‐ and tripeptides, to be used as a health food. This product was found to have anti‐inflammatory property. It is possible that the antiproliferative effects of soy protein on breast cancer in rats may be mediated by its hydrolysis in the small intestine to small peptides. Hence, we hypothesized that the beneficial effect of soy protein in breast cancer development may be mediated by its hydrolysis product as peptides. Hinute‐AM was solubilized in 0.1% FA and analyzed by LC‐MS/MS. The data analysis has revealed the presence of five peptides (VPY, EL, IQ, LQ and YG) in Hinute‐AM. Out of these, three synthetic peptides (YG, LQ and IQ) were tested for their effects on proliferation of three normal (12A, 12F and 184A1) and four breast cancer cells (HCC1806, MCF 7, BT‐549 and MDA‐MB‐231). All peptides had significant antiproliferative effects in all breast cancer cell lines. However, the major effect was with YG and in particular in HCC1806 cell line. We reported earlier that expression of TSPO (translocator protein) is higher in breast cancer cells than normal breast cells. PCNA is also regarded as good cell proliferation marker. In this experiment, we studied the effects of YG on TSPO and PCNA expression in normal and three breast cancer cells (MCF‐ 7, BT‐549 and HCC‐1806 by immunohistochemistry. Results indicated that YG down‐regulated the expression of both TSPO and PCNA in all cancer cells. Furthermore, the binding capacity of 3H Ro5‐4864 to the TSPO receptors was significantly reduced by both YG and LQ. The mechanism by which YG and LQ inhibited the proliferation of breast cancer cell lines is under investigation. (Supported by grants from Fuji Oil Company, Osaka, Japan and NIH 5U54MD007593)

Support or Funding Information

Supported by grants from Fuji Oil Company, Osaka, Japan and NIH 5U54MD007593

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FOXA2 PROMOTES PROSTATE CANCER BONE COLONIZATION
Authors: connelly zachary m. , Yang Shu , Orr A. Wayne , Yu Xiuping
Published: June 2021

Background

It is estimated that 26,000 men will die from prostate cancer (PCa) in 2017. Androgen deprivation therapy (ADT) is the gold standard treatment for advanced PCa. Initially, patients respond to treatment, however, these tumors almost invariably progress to castrate‐resistant PCa, for which there is no cure. Most PCa patients who fail ADT develop metastasis and preferentially relocate to the bones. Bone metastasis results in significant morbidity and mortality as the average time to death is approximately 3–5 years with no treatment available. Understanding the mechanisms by which PCa grow in the bone is critical for the development of novel therapeutics to treat and decrease tumor‐mediated bone destruction.

Methods

Gene expression profiling studies have found that FOXA2, a forkhead transcription factor that is expressed in embryonic prostate and neuroendocrine PCa, is present in a subset of metastatic PCa specimens. Our preliminary study found FOXA2 expression in a sample set of human PCa bone metastases, suggesting an involvement of FOXA2 in human PCa bone metastases. Also, we found high levels of FOXA2 in aggressive PCa PC3 cells, but not in PCa LNCaP cells. PC3 cells generate osteolytic lesions in bone, whereas LNCaP cells minimally grow following bone inoculation. To establish FOXA2's role in promoting PCa metastasis, FOXA2 was stably knocked down in PC3 cells and overexpressed in LNCaP cells.

Results

We found that FOXA2 knockdown in PC3 cells resulted in a significant decrease in PC3 mediated in vivo bone destruction following intra‐tibial injection. To understand how FOXA2 is facilitating these changes, we examined the expression of integrins and observed that FOXA2 knockdown decreased the expression of collagen‐binding integrins α1 and αv in PC3 cells. Furthermore, we found FOXA2 knockdown decreased PC3 cells' adhesion and spreading on collagen I (a major component of bone ECM) coated surfaces. The Foxa2‐controlled expression of integrins α1 and αv and the resulting changes in the adherence and spreading would provide a mechanism for PCa cells colonize bone and initiate the bone‐destruction cycle. Additionally, FOXA2 knockdown in PC3 cells resulted in a significant decrease in expression of parathyroid hormone‐related protein (PTHrP), a bone remodeling‐associated protein. We observed that PTHrP mRNA was decreased in FOXA2 knockdown cells and protein in FOXA2 knockdown subcutaneous grafts. When PC3 FOXA2 knockdown cells were co‐cultured with osteoblast and osteoclast, osteoclast markers were decreased.

Conclusions

Taken together, FOXA2 plays a major role in facilitating PCa's ability to colonize bone, and further, promote osteoclast activation.

Support or Funding Information

Feist‐Weiller Cancer Center/Foundation of Legacy Funds The Office of Research SEED awards

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Receptor Tyrosine Kinase Signalling in the Absence of Kinase Activity and Cancer of Non‐Genetic Origin
Authors: Ladbury John
Published: June 2021

FGFR2‐expressing cancer cells with low concentrations of the adaptor protein Grb2 show high prevalence for metastatic outcome. In non‐stimulated cells the SH3 domain (and not the SH2 domain(s)) of Plcγ1 directly competes for a proline‐rich binding site at the very C‐terminus of FGFR2 with the C‐terminal SH3 domain of Grb2. Reduction of Grb2 concentration permits access of Plcγ1 to the receptor. Recruitment of Plcγ1 in this way is sufficient to up‐regulate phospholipase activity. This results in increased cell proliferation, motility and promotion of cell invasive behaviour in the absence of extracellular receptor stimulation. Therefore tumour growth and metastatic outcome can be dictated by the constitutive competition between Grb2 and Plcγ1 for the phosphorylation‐independent binding site on FGFR2. Since the majority of receptor tyrosine kinases have proline‐rich sequences in their C‐termini, the possibility of a second tier of signal transduction in the absence of growth factor stimulation, or kinase‐activating mutations emerges – leading to cancer of non‐genetic origin. Further examples of this second tier of signalling have been investigated and mechanistic detail of how protein concentration fluctuations in response to cellular stress can lead to pathogenesis has been established.

Support or Funding Information

This work is funded by CRUK

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 sphingosine kinase 2 in promoting multiple myeloma cell invasive growth
Authors: Zhang Hannah , Zhao Derrick , Li Xiaojiaoyang , Liu Runping , Wang Xuan , Zhou Huiping
Published: June 2021

Multiple myeloma (MM) is a heterogeneous disease with a prognosis that varies with patient genetics, disease burden, tumor biology, and treatments. Effective therapy for high risk MM is currently unavailable due to the limited understanding of its pathophysiology. Sphingosine 1‐phosphate (S1P) has been identified as an important signaling molecule involved in regulating various cancer cell growths. Two protein kinases responsible for S1P synthesis, sphingosine kinase 1 and 2 (SphK1 and SphK2), have been identified. During the last decade, numerous studies have shown that SphK1 plays a critical role in promoting various cancer cell growths. However, only limited attention has been paid to SphK2. Our recent studies reported that activation of SphK2 is linked to invasive growth of cholangiocarcinoma. However, the role of SphK2 in MM cell growth is not clear and is the focus of this study.

Methods

Studies were conducted in the human MM cell lines, KMS‐11, KMS‐12BM, KMS‐20, KMS‐21BM, KMS‐26, KMS‐28BM, KMS‐34, MOLP‐8, LP‐1, MM.1S. The expression of SphK1 and SphK2 was determined by real‐time RT‐PCR and Western blot analysis and confirmed by DNA sequencing. The SphK2 inhibitor, K145, and gene‐specific shRNA were used to inhibit SphK2 activation and expression. Cell proliferation was determined using a CCK‐8 kit. Cell apoptosis was analyzed by Annexin V‐FITC/PI staining followed by flow cytometry.

Results

The expression level of SphK2 in all tested MM cell lines was significantly higher than SphK1. S1P significantly induced proliferation and growth of MM cells, which was inhibited by K145 as well as gene specific shRNA targeting SphK2. The SphK2 expression level is correlated to invasiveness of MM cell growth and its sensitivity to K145.

Discussion/Conclusion

SphK1 and SphK2 are all expressed in human MM cancer cells. However, the SphK2 expression level is much higher than SphK1. In this study, we were able to show that SphK2 plays a critical role in S1P‐mediated invasive growth of MM cells. The results in this study suggest that targeting SphK2 may represent a novel therapy for MM.

Support or Funding Information

This work was partially supported by National Institutes of Health Grant R01 DK104893 and R01DK‐057543; VA Merit Award I01BX001390; National Natural Science Foundation of China Grants 81070245 and 81270489.

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