bluuebunny/crossref_metadata_2025 huggingface dataset:
Insight into Parkinson's Disease from Yeasts: Combined impact of covalent modifications & familial mutations on α‐synuclein
Authors: Ganev Yoan Petrov , Thomas Rosemary , Roman Alexandra , Mwale Chisomo , Mountain Joseph , Balaram Ariane , Marshall Morgan , DebBurman Shubhik
Published: June 2021

Parkinson's Disease (PD) is a neurodegenerative disorder linked to the loss of dopaminergic neurons in the midbrain. A key pathological marker of PD is the presence of Lewy bodies, which are mainly composed of misfolded α‐synuclein protein. α‐synuclein is a highly post‐translationally modified protein. While phosphorylation and nitration of α‐synuclein are well studied in the context of PD pathology, less is known about sumoylation, which is proposed to be neuroprotective based on limited studies. The majority of sumoylation takes place on the lysine‐96 and lysine‐102 sites of α‐synuclein, and it increases the protein's solubility. The goal of this research was to better understand the role of sumoylation in regulating α‐synuclein toxicity, and we performed four studies towards it. First, we evaluated the effects of blocking sumoylation on α‐synuclein in the well‐established budding yeast model for PD and found that α‐synuclein becomes more aggregated, gains toxicity, and loses localization at the plasma membrane. Second, we evaluated the effects of altering sumoylation pathways by using yeast strains with reduced (ulp1ts) or excessive sumoylation (smt3ts), and found that α‐synuclein aggregates more with reduced sumoylation, but becomes less toxic with increased sumoylation. Third, we asked how altering phosphorylation of α‐synuclein would alter sumoylation's protective role and found that blocking phosphorylation (in α‐synuclein already blocking sumoylation) reduced the protein's toxicity. Finally, we began evaluating whether blocking sumoylation and altering phosphorylation on familial PD mutant versions of α‐synuclein would exacerbate its toxicity. We found preliminary evidence that the toxicity of the A53T mutation increases when sumoylation is blocked. In the future, we will conduct further studies to understand how sumoylation affects other variants and modifications of α‐synuclein.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quantifying the Aggregation Propensity of IAPP from Diabetic and Nondiabetic Species
Authors: Moffet David , Palato Larry , Pilcher Shannon , Rinauro Dillon , Menefee Kate , Tun Angela , Jauregui Betssy , Shapiro Sarah , Nossiff Olivia , Nguyen Viviane
Published: June 2021

The aggregation of the 37‐amino acid polypeptide human Islet Amyloid Polypeptide (hIAPP, amylin), as either insoluble amyloid or as small oligomers, appears to play a direct role in the death of pancreatic β‐islet cells in type 2 diabetes. It is known that several organisms express non‐amyloidogenic variants of IAPP (such as rat and mouse) and are not known to develop diabetes naturally. Conversely, several organisms express highly amyloidogenic variants of IAPP (such as human, cat and primates) and are known to develop diabetes. Despite the significant amount of genetic information available, no comprehensive study has been conducted to directly correlate IAPP aggregation potential to the propensity to develop diabetes within the animal kingdom. In this work, we will compare the aggregation potential of naturally occurring IAPP variants from organisms known to, or known not to, develop diabetes. IAPP peptide variants were chemically synthesized. Each variant was tested for its amyloid aggregation potential using Circular Dichroism and Atomic Force Microscopy.

Support or Funding Information

National Institute of Health (Grant#: 1R15DK112172)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Defining the fumarase water binding site
Authors: Weaver Todd Michael
Published: June 2021

Fumarase C, a class II family member, catalyzes the nearly catalytically perfect reversible conversion of fumarate to S‐malate as part of the Krebs Cycle. Despite decades of research and high quality structural information the exact mechanism for diffusion limited catalysis remains elusive. In order to more fully understand the contribution of various active site residues to the fumarase‐catalyzed reaction, a series of crystal structures and kinetic parameters for a series of active site fumarase variants have been determined. The first set of variants targeted the water 26 (W26) binding site formed via Ser98, Thr100, Asn141 and His188. Alterations at the water‐binding site, termed the W26 cluster, affected the Km and kcat. The structural data for each W26 cluster replacement notes only slight shifts in this structural water location with maximal movement of 4.9 Å in the H188A active site leading to an overall 3500‐fold reduction in activity. The second set of variants replaced the highly conserved glutamic acid at position 331 with either an aspartic acid or a glutamine residue. Glu331, contained within the fumarase signature sequence, forms a shortened hydrogen bond to stabilize His188 just behind the W26 cluster. The Glu331 to aspartic acid or glutamine substitution create significant reductions in kcat and limited effect on Km values even though there are few structural changes in the position of W26. Based upon the structural and kinetic data we propose the following (1) W26 provides a model for both the S‐malate 2‐[OH] binding site during the S‐malate dehydration reaction and the water binding site during the fumarate hydration reaction, (2) Asn141 and Glu331 facilitate Nδ1‐H to Nɛ2‐H tautomeric interconversion of His188 and (3) His188 acts as the catalytic acid to protonate the S‐malate 2‐[OH] during the S‐malate à fumarate dehydration.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kinetic Characterization and Chemotherapeutic Relevant Inhibition of Human Malate Dehydrogenase 1 and 2.
Authors: Wardenfelt Stacey , Dwyer Tim
Published: June 2021

Docetaxel and doxorubicin are chemotherapeutic agents used to fight cancers ranging from prostate cancer to uterine cancer to leukemia to soft tissue sarcomas. However, patients can often develop resistance to docetaxel, and doxorubicin can be cardiotoxic which limits its usefulness. Recent data suggest that both of these problems are in part caused by overexpression of the mitochondrial malate dehydrogenase (MDH2) protein in tumor cells and that inhibitors of this enzyme, such as mebendazole and visnagin, could greatly increase the efficacy of docetaxel and doxorubicin. Studies have yet to address the how these inhibitors affect the cytoplasmic isoform of malate dehydrogenase (MDH1) and the possible side effects the inhibition of this non‐target enzyme could have. One of the functions of MDH1 is to help in gluconeogenesis. Malignant cells are often more dependent on glycolysis for energy generation than normal cells. Since gluconeogenesis is antithetic to glycolysis, inhibiting an enzyme that helps in gluconeogenesis (such as MDH1) could promote glycolysis and, in essence, enhance the growth of tumor cells. The current study has utilized the pMAL expression system (New England Biolabs) to express human MDH1 and MDH2. The expressed enzymes have been kinetically characterized in the absence and presence of known and new inhibitors with the idea of finding an inhibitor that is specific for MDH2.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optimization of the Enzymatic Synthesis of UDP‐Xylose and Characterization of Enzymes Involved
Authors: Cook Matthew , Culbertson Alan , Zabotina Olga
Published: June 2021

The plant cell wall is of interest for agriculture and biofuel production; understanding the biosynthesis of plant cell walls is critical for improving the viability of biorenewable energy as a replacement for traditional energy sources. Xyloglucan is an important cell wall constituent and has high degrees of xylosylation. However, studies of xylosylation of hemicelluloses and other biomolecules are hampered, partially due to the low supply of the primary substrate for xylosylation, UDP‐xylose (UDPX). The goal of this project has been to optimize the known biological pathway for UDPX synthesis for laboratory use. The two‐enzyme UDPX biosynthesis pathway produces UDPX from UDP‐glucose (UDPG), a more readily available substrate chemical. The project also characterized enzymatic properties of the enzymes UDP‐glucose dehydrogenase (Ugd) and UDP‐xylose synthase (Uxs), which perform the catalytic activities in the pathway of interest. These enzyme homologs have not been characterized previously.

Ugd and Uxs were expressed and purified using the plasmids prepared in Dr. Adam Barb's lab. While homologs of Ugd and Uxs have been partially characterized before, neither has been studied to the degree required for this project and the specific homologs used herein have never been reported. Enzymatic assays were used to determine the optimum conditions for each enzyme and spectrophotometry and high‐performance liquid chromatography were used to measure the products of reactions. Biologically relevant conditions including pH, salt content, reducing agent content, presence of metal ions, et cetera were optimized. Subsequently, methods of combining the reaction systems have been attempted. Due to the significantly different pH optimums of the two enzymes and the generation of inhibitory compounds, the synthesis of UDPX cannot be performed as a one‐pot reaction and will require two independent, consecutive reactions. Additionally, kinetics data collected has provided interesting insights into the enzymatic properties of Ugd and Uxs. Thus, it was shown that Ugd experiences substrate inhibition, but this substrate inhibition can be relieved by the addition of ATP, despite ATP not being involved in the reaction.

The primary goal of the project has been the efficient synthesis of UDPX from UDPG. To that end, the individual assay conditions have been improved to over 90% conversion efficiency for both enzymes, individually. The conversion of UDPG to UDPX is now being performed in a consecutive reaction series and the efficiency of this reaction series is being improved.

Support or Funding Information

This work was supported by NSF‐MCB grant #1121163

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeting DXP Synthase using TPP Mimics
Authors: Alsalahi Amera , Girardi Peter , Callahan Kevin , Sridhar Anand
Published: June 2021

A global emergence of antibiotic‐resistant bacteria, largely due to the misuse and over use of antibiotics, has led to an increase in demand for the development of new antibacterial agents. In order to develop new antibiotics, researchers need to identify new targets that have high specificity for certain bacterial pathways. The enzyme D‐xylulose 5‐phosphate (DXP) synthase catalyzes the first step in the MEP (methylerythritolphosphate) pathway in human pathogens, which produces the biological molecules necessary to regulate numerous cellular functions, including central metabolism. Inhibition of DXP synthase would significantly reduce the rate of DXP production and the MEP pathway and thus is an ideal candidate for antibiotic development. DXP synthase requires the cofactor, thiamin pyrophosphate (TPP) for its activity. Preventing TPP from complexing with DXP synthase will inhibit the activity of the enzyme and consequently inhibit the MEP pathway. To test the efficacy of DXP synthase inhibitors both DXP synthase and the sequential enzyme of the MEP pathway, DXP reductoisomerase (IspC), were purified from E.coli using nickel affinity chromatography. Conversion from DXP to 2‐C‐Methyl‐D‐erythritol 4‐phosphate (MEP) by IspC coincides with NADPH oxidation that can be measured using spectroscopic techniques. The establishment of this in vitro system will allow us to test the efficacy novel DXP synthase inhibitors.

Support or Funding Information

This project was funded by SJFC internal 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.
Nano‐Targeted Kinase Inhibitor Treatment of KRAS Mutant Lung Cancer
Authors: Sridharan Ramya , Shah Janki , Shamay Yosef , Heller Daniel
Published: June 2021

Lung cancer is the leading cause of mortality in the US, affecting 160,000 patients annually with a five year survival of 17%. The majority of lung cancers harbor KRAS mutations that over‐activate the RAS kinase in the RAS‐MAPK signaling pathway, RAS‐RAF‐MEK‐ERK. This leads to increased pathway activity and increased cell division and tumor growth. Kinase inhibitors (KIs), which target key effectors of cancer signaling pathways, constitute a major strategy to treat KRAS‐driven lung cancer. Although RAS itself is difficult to drug with KIs due to the lack of binding pockets, KIs inhibiting downstream MEK kinases of the RAS‐MAPK pathway have shown to be most effective in vitro. However, MEK inhibitors suppress signaling in non‐cancer cells, so their dosing is limited by toxicity and results in a narrow therapeutic index. Additionally, the anti‐tumor effects of MEK inhibitors in KRAS‐driven lung cancer are further diminished by the activation of compensatory pathways, resulting in drug resistance and tumor resurgence. Although the co‐administration of a second KI can mitigate this resistance, many such combinations of KIs are highly toxic.

We recently developed a new nanomedicine platform to package kinase inhibitors with high drug loading. These indocyanine nanoparticles (INPs) alter the systemic biodistribution of the KI to improve the drug's therapeutic index via tumor targeting. The INPs target caveolin receptors overexpressed on cancer endothelium to increase the local concentration of kinase inhibitor at the tumor site and reduce the systemic exposure of non‐cancerous tissues. Thus, the selective targeting decreases the toxicity of systemic KI exposure to concomitantly improve the therapeutic index.

Support or Funding Information

This work was supported in part by the NIH New Innovator Award (DP2‐HD075698), the Cancer Center Support Grant (P30 CA008748), the Pershing Square Sohn Cancer Research Alliance, the Honorable Tina Brozman Foundation for Ovarian Cancer Research, the Expect Miracles Foundation ‐ Financial Services Against Cancer, the Anna Fuller Fund, the Louis V. Gerstner Jr. Young Investigator's Fund, the Frank A. Howard Scholars Program, Cycle for Survival, the Alan and Sandra Gerry Metastasis Research Initiative, Mr. William H. Goodwin and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research, the Experimental Therapeutics Center, the Imaging & Radiation Sciences Program, and the Center for Molecular Imaging and Nanotechnology of Memorial Sloan Kettering Cancer.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Imaging Exosomes using Super Resolution Microscopy
Authors: Chen Chen , Zong Shenfei , Wang Zhuyuan , Cui Yiping
Published: June 2021

Exosomes are small membrane vesicles originated from late endosomal compartments of cells with a diameter ranging from 30–100 nm. Studies have demonstrated that exosomal miRNAs can influence the biological processes of recipient cells. Therefore, direct imaging and tracking of exosomes and exosomal miRNAs in recipient cells are essential for exosome‐related functional analysis. However, the moderate spatial resolution of conventional fluorescence microscope limits the precise localization of exosomes considering their relatively small size (<100 nm). Here, we took advantage of single molecule localization microscopy (SMLM) to realize the visualization and intracellular functional analysis of exosomes and exosomal miRNAs in recipient cells.

First, the multi‐color SMLM imaging of exosomal membrane receptors (CD63 and HER2) and exosomal miRNAs (mir‐21 and mir‐31) were accomplished with more reliable structural information and localization accuracy. Exosomes taken up by normal cells can be more precisely visualized through SMLM. In our research, the motion of exosomes within the intercellular filamentous structures was observed successfully, indicating one kind of the intercellular communication pathways. According to the dual‐color SMLM imaging of exosomes and lysosomes, we confirmed that exosomes can be transported into lysosomes for further degradation after binding to recipient cells. Moreover, as one kind of the natural carriers of the cellular cargoes, exosomes can encapsulate mir‐21 inside to prevent enzyme degradation during transferring and then release them into recipient cells. Specifically, these released exosomal mir‐21 were able to target the 3'UTR of TPM1 mRNA, which can repress the expression of the target gene.

These findings can provide new insights into the pathway of intercellular communications and affirm that SMLM is a powerful technique for the exploration of exosome‐mediated cancer metastasis, as well as the role of exosomal miRNAs in gene regulation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cryogenic Grinding and 3D Printing Techniques for Establishing “Disperse and Absorb” Brick‐Type Constructs of Food Materials
Authors: Rhee Jin‐Kyu
Published: June 2021

The potentialities of 3D printing technology are discussed from technical and research‐oriented perspectives for industrial manufacturing of a variety of food products. Currently, 3D printing technology has been advanced to enable us to process or cook innovative foods. However, food‐based materials for 3D printing are still limited in terms of eating qualities, nutritional values and functionality as well as industrial production. Therefore, this uprising issue on alternative food processing techniques especially focused on the exploration of new food materials combined with these 3D printing technologies needs to be re‐spotlighted, and then solved to pave the way to this innovative and sensational area of investigation with more accessibility. In this work, fabrication parameters were characterized and optimized to improve printability, quality and efficiency of manufacturing foods. First, carbohydrates/protein‐based food materials were cryo‐powdered to render an amorphous shaped‐ micronized‐ materials with characterization. Subsequently, modification of structure with this micro‐sized food materials has been re‐crystallized by controlling water contents and heat. Then, bonding materials has been formulated and jetted onto the carbohydrates/protein‐based food powders to be polymerized. Construction of cohesive micro‐sized geometry through combining materials with controllable parameters was optimized by the responsive surface methodology. This technology has a potential to design the exterior of foods and internal microstructure to give texture with controlled body‐absorption.

Support or Funding Information

This research was supported in part by the High Value‐added Food Technology Development Program, the Ministry of Agriculture, Food and Rural Affairs (MAFRA), Republic of Korea (316058‐3, 315063‐3), the Functional Districts of the Science Belt support program, Ministry of Science, ICT and Future Planning, Republic of Korea (2017K000017), and the Ewha Womans University Research Grant of 2017.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Considerations for Western Blot Normalization Techniques
Authors: Oh Kenneth , Dreskin Elizabeth Jordan , Liu Ning , Hammond Matthew , Kollmann Franziska , Posch Anton
Published: June 2021

Western blotting is a well‐established method to specifically measure the relative quantities of individual proteins in complex biological samples. However, data quality may be affected by technical and biological variation. The application of data normalization strategies is strongly recommended to compensate for these non‐sample related variations in signal intensity. Here, we compare the power of two different data normalization strategies, namely housekeeping protein (HKP) and total protein (TP) normalization based on Stain‐free technology to offer an orientation in selecting the appropriate data normalization tool. In brief, HeLa cell lysates were separated with SDS‐PAGE Stain‐free gels and blotted onto a PVDF membrane. Stain‐free imaging after activation with UV was applied to obtain a total protein blot signal. The blot was then treated to the appropriate immuno‐probing steps and the intensity of the target protein ATG7 was recorded by chemiluminescence. In order to illustrate the differences of the two techniques, the membrane was subsequently immuno‐probed for GAPDH as a typical example for housekeeping protein data normalization. Both data sets were used for data normalization of the target protein ATG7. Our results revealed that total protein staining through Stain‐free technology shows less technical variation than HKP recording as represented by GADPH. Additionally, the use of housekeeping proteins as data normalization tool is restricted in its general applicability and may not be compatible with the strict publishing journal guidelines.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An Aptamer‐Based Approach to Assess the Human Plasma Proteome for Pre‐Analytical Variability
Authors: Daniels Jaclyn R. , Cao Zhijun , Maisha Mackean , Schnackenberg Laura K. , Sun Jinchun , Pence Lisa , Schmitt Thomas C. , Kamlage Beate , Beger Richard D. , Yu Li‐Rong
Published: June 2021

The quality of clinical samples is of utmost importance when conducting biomedical research with OMICS‐based assays as the variability introduced before sample analysis is a potential confounder in the results. In this study, we used the aptamer‐based SOMAscan® proteomic technology to characterize pre‐analytical variability in human EDTA plasma. The goal of the study was to investigate how variation in the pre‐analytical steps during processing of whole blood to plasma impacts the plasma proteome. Whole blood was obtained from apparently healthy individuals and was collected in a randomized sequence of six EDTA tubes. The pre‐analytical variables included prolongation of the processing start time, storage temperature of whole blood or plasmaprior to and after processing, and variation in applied centrifugal force. The SOMAscan® assay was performed to measure the relative abundance of 1305 proteins. To determine the effects of variations inpre‐analytical processing, the data was statistically analyzed and the results were used to identify significant changes in protein abundance under each study condition relative to the control condition (samples processed without delay and immediately stored at −80°C). Both low centrifugation force(1300 × g compared to 2500 × g) and blood storage at 0°C for 6 hours resulted in the most significant changes in protein abundance (200 and 148 proteins, respectively, fold change ≥ 1.2, P < 0.05, and FDR < 0.05), while storing immediately processed plasma for 24 hours at 4°C yielded the least changes (28 proteins). The results indicate that blood samples should be immediately processed to plasma and frozen in −80°C without delay in order to preserve sample integrity and to minimize any proteome abundance alterations. The results also provide important information on the discovery of protein abundance changes due to pre‐analytical variability that can be useful in performing effective quality control assessments.

Support or Funding Information

This study was supported with funds from the National Center for Toxicological Research, Food & Drug Administration, Jefferson, Arkansas.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bacterial Viruses Organize Subcellular Environments to Mediate Heterogeneous Development
Authors: Trinh Jimmy Tri , Zeng Lanying
Published: June 2021

Spatial organization underpins biological processes in lifeforms. For complex organisms, body parts must develop properly in space, and within the cells making up these organisms, membranes separate organelles with different functions. Simpler organisms, like bacteria, lack intracellular membranes, but possess alternate strategies for organization, where certain proteins are confined to specific cellular locations to consolidate their functions. Viruses, among the simplest lifeforms, have also been reported to display organized development, further underscoring the essentiality of structure in biology.

Bacterial viruses, known as bacteriophages or phages, are utilized as model systems for studying advanced cellular functions. Phage lambda infects Escherichia coli and propagates via distinct pathways, either lysis or lysogeny, where this fate‐selection is a paradigm for cellular decision‐making. Recent efforts probing decision‐making at the subcellular level have discovered that phages behave as individual interacting intracellular entities when processing decisions, generating considerable insights into how complex decision‐making may occur. However, little is known about this phage's spatial development within its host and how spatial organization might affect cellular decision‐making.

Here, we characterize how phage lambda develops in subcellular space, from infection through lysis, using multiple fluorescence reporters targeting phage DNA replication, essential host resources, phage transcription, and phage assembly. We observe that these processes coalesce into distinct areas of the host cell during infection to coordinate phage development, reminiscent of virus factories, where multiple factories may be present in single cells. Remarkably, different intracellular factories quantitatively diverge at the viral DNA, mRNA and protein levels, suggesting the existence of bona‐fide microenvironments within cells. We propose that individual viruses drive the formation of disparate territories, implying that phages may sense separate environments during decision‐making and development. The conclusions from this work may shed significant light on the detailed molecular mechanisms of cell‐fate bifurcation.

Support or Funding Information

This work was supported by the National Institutes of Health (R01GM107597).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RXRA is a direct target gene of miR‐506‐3p that regulates oncogene MYCN expression and cell differentiation in neuroblastoma
Authors: Shelton Spencer Dewayne , Zhao Zhenze , Du Liqin
Published: June 2021

Neuroblastoma is an aggressive childhood cancer that arises from neural precursor cells that fail to differentiate. In 20% of high‐risk neuroblastoma cases oncogene MYCN is overexpressed. We previously identified that miR‐506‐3p is a strong repressor of MYCN expression as well as an inducer of neuroblastoma differentiation. However, the 3'UTR of MYCN mRNA lacks the target site of miR‐506‐3p, suggesting that miR‐506‐3p regulates MYCN expression through an indirect pathway. Combining gene expression array data and informatics analyses, we identified 11 candidate genes that are direct targets of miR‐506‐3p and potentially regulate MYCN expression. The candidate genes were silenced and screened for decreased MYCN expression and increased neuroblastoma neurite outgrowth – the morphological differentiation marker of neuroblastoma cells. Interestingly, retinoid X receptor alpha (RXRA) was the only gene to exhibit significant downregulation of MYCN expression and induced the most dramatic neurite outgrowth. We then examined the RXRA protein expression during retinoic acid treatment and demonstrated that RXRA protein level is significantly downregulated in retinoic acid treated cells. Upon investigation of neuroblastoma patient databases, we found that high risk and MYCN amplified patients that exhibited higher RXRA mRNA levels had lower overall and relapse free survival statistics. Altogether, these results suggest that RXRA functions as an oncogene through promoting MYCN expression and repressing cellular differentiation in neuroblastoma. Further investigation is ongoing to elucidate the molecular pathways that mediate the oncogenic function of RXRA in neuroblastoma 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.
Investigation of Domain Responsible for Calcineurin B Homologous Protein (CHP) Isoform Specific Function
Authors: Davis Shane , Wallert Mark , Provost Joseph
Published: June 2021

Mammalian cells ubiquitously express the Sodium‐Hydrogen Exchanger (NHE1) a membrane transporter responsible for intracellular pHi, motility and proliferation. Calcineurin B Homologous Protein (CHP) regulates activation and kinetics of the NHE1. CHP has two isoforms, CHP1 and CHP2, whose physiological function remains unclear but both have a unique role in nascent tumor survival. There is a signature domain in the sequence of an otherwise homologous structure between CHP1 and CHP2 that we believe is responsible for CHP isoform specific function, including regulation of NHE1. The purpose of this study is to understand how the these domains of CHP1 and CHP2 confer to the properties of the CHP, particularly cell proliferation and migration. To identify possible differences of structural conformation that could be related to function of each CHP isoform we exchanged the (aa‐aa ofr CHP1 and aa‐aa of CHP2 using gibson cloning. Gibson cloning allows us to manipulate fragments created by PCR to make mutants of our wild‐types. These constructs were then transiently transformed into cells and changes in proliferation and migration analyzed compared to wild‐type expressing cells. We hypothesize that there is an interaction between CHP1 and CHP2 that allow for their individual absence or presence to affect the proliferation and motility function in lung carcinoma.

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 hydrolytically activated, oxygen‐generating biomaterials to enhance drug efficacy on cultured ovarian cancer cells
Authors: Clark Matthew Paul , Kraus Aileen , Higgins Jillian , Gallant Benjamin , Arruda Dominic , McTigue Christie , Li Xiaofei , Guo Hao , Chen Hongduo , Cui Yali , Di Wen , Wan Yinsheng
Published: June 2021

Ovarian cancer remains the most prevalent type in gynecological cancer, with drug resistance still being a challenge. Our previous studies have demonstrated that ovarian cancer cells are resilient due to their active EGFR and AKT/mTOR pathways. Also, abundant data suggests that similar to other cancer types, ovarian cancer cells grow and proliferate remarkably well under hypoxia situation. We hypothesize that the application of oxygen to ovarian cancer cells may render unfavorable conditions, thus leading to the enhanced cell death, when combined with chemotherapy drugs. Recent studies have shown that polydimethylsiloxane (PDMS)‐encapsulated calcium peroxide, or PDMS‐CaO2, potently deliver oxygen and eliminate hypoxia‐induced cell dysfunction and cell death in normal pancreatic cells and other types of normal cells. In this study, we followed the published protocol, produced PDMS‐CaO2 disks and tested in vitro the effects of released oxygen on cultured ovarian cancer cells (CaOV3 cells). The results showed that our manufactured disks sustainably release O2 in a time dependent manner. MTT assay results indicated that PDMS‐CaO2 disks induce cell death in an oxygen dependent manner, compared to PDMS disks only. Combination of inhibitors of EGFR, MEK/ERK and AKT/mTOR with PDMS‐CaO2 disks increases cell death. Interestingly, PDMS disks significantly enhance the effects of cisplatin, oxaliplatin and doxorubicin, but not that of paclitaxel or taxol. PDMS‐CaO2 disks inhibit exosome inhibitor GW4869 induced cellular proliferation. Also, those disks significantly augment ionophore monensin A (for Na+/H+ antiporter), and calcium ionophore A23187‐induced cell death. Confocal microscope data showed that PDMS‐CaO2 disks alter mitochondria activities. Collectively, our data suggests that PDMS‐CaO2 disks releasing O2 and inducing cell death may affect cell membrane ion channels and mitochondria activities. The combination of cancer drugs or ionophores or EGFR/MEK/AKT/mTOR inhibitors with PDMS‐O2 disks may provide novel approaches for ovarian cancer clinical management.

Support or Funding Information

NIH

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Impacts of Hypoxia on Na+/H+ Exchanger Isoform 1 and Calcineurin B Homologous Protein Isoform 2 Expression in Non‐small Cell Lung Cancer
Authors: Wallert Clarice H. , Provost Joseph J. , Wallert Mark
Published: June 2021

The Na+‐ H+ exchanger isoform 1 (NHE1) is a key regulator of cell proliferation, migration and invasion in cells from a variety of solid tumors. The calcineurin B homologous proteins (CHP1 and CHP2) appear to be essential cofactors to support NHE1 function. CHP1 appears to be expressed ubiquitously in healthy tissue, while CHP2 is predominantly expressed in tumor cells. While both CHP isoforms are highly homologous and bind in nearly identical regions on NHE1, each has been identified to have a distinct impact on NHE1 function. Our research has demonstrated an elevation in CHP2 expression in tumor samples from patients with either adenocarcinoma or squamous cell carcinoma of the lung as well as in a broad range of NSCLC cell lines. To evaluate the role of CHP2 in NSCLC cells we used NCI‐H1299, a carcinoma cell line, as the parent cell line and developed two new cell lines. One of these cell lines lacked CHP2 expression (H1299 CHP2KD) and the other lacked NHE1 expression (H1299 NHE1KD). We hypothesized that CHP2 expression enhances cell survival and the tumorigenic potential of these cells. We evaluated the impact of a low serum and hypoxic microenvironment on CHP2 expression and determined CHP2 levels increased when H1299 cells are grown in 0.5% serum and 1% O2 compared to cells grown in 10% serum and 21% O2 (normal conditions) after 24 and 48 hours; however, at approximately 72 and 96 hours, CHP2 expression in the same conditions decreased while NHE1 expression increased. CHP2 may be involved in initial survival mechanisms during the time NHE1 expression is increasing to further allow for cancer progression. To evaluate the role of CHP2 and NHE1 biologically, proliferation and migration were assessed in all conditions. Proliferation and migration rates in normal conditions for H1299 CHP2KD cells were approximately 50% of H1299 cells. When these experiments were performed in low serum and hypoxic conditions, the proliferation and migration rates of the H1299 cells was reduced to about 50% of that seen in normal conditions, while the rates of proliferation and migration in H1299 CHP2KD was reduced to a rate equivalent to the H1299 NHE1KD cells. These data suggest a major role for CHP2 expression in the development and progression of non‐small cell lung cancer.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ascites tumor microenvironment and chemoresistance in ovarian cancer
Authors: Kim Soochi
Published: June 2021

Ovarian cancer is inherently silent; most patients are diagnosed in late stage of the disease progression. The progression and resistance to chemotherapy in ovarian cancer patients is closely associated with aberrant accumulation of fluid in the peritoneal cavity, called ascites formation. Malignant ascites constitutes a unique tumor microenvironment providing a physical structure for the accumulation of both pro‐tumorigenic and anti‐tumorigenic factors, each of which cumulatively favor cancer cell proliferation, metastasis and chemoresistance. Almost all patients with recurrent ovarian cancer are presented with ascites and have acquired resistance to chemotherapy. However, underlying mechanism of ascites induced acquired resistance to chemotherapy have not been fully understood yet. We used patient derived ascites to delineate the role of ascites in acquired chemoresistance in ovarian cancer. Of those many soluble factors, we focused on the levels of cholesterol in malignant ascites. We found that cholesterol levels are highly elevated in malignant ascites to that of benign peritoneal fluid from ovarian cyst patients. Treatment of cholesterol significantly induced resistance to cisplatin and paclitaxel via increased expression of drug resistance proteins, ABCG2 and MDR1, together with upregulation of LXRα/β. Moreover, LXRα/β expression was positively correlated with MDR1 but not with ABCG2 expression in ovarian cancer patient‐derived ascites cells. The importance of cholesterol induced LXRα/β in acquired resistance were further confirmed by silencing LXRα/β. In addition, elevated cholesterol in malignant ascites was associated with shorter recurrence free survival in ovarian cancer patients. Collectively, these results suggest that cholesterol in ovarian cancer patient derived ascites causes resistance to cisplatin through upregulation of MDR1 via activation of LXRα/β in ovarian cancer. Targeting the pathological interaction between cancer cell and ascites tumor microenvironment may be an efficient strategy to overcome acquired resistance in ovarian cancer patients.

Support or Funding Information

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HA17C0037, HI16C2037) and grants from Seoul National University College of Medicine (800‐20150304) and the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea (1720110).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhancement Natural Killer Cell Activity of Fucoidan in Lung Metastasis in vivo Model
Authors: Kim Jinhye , Kim Da Hyun , Chun Su‐Hyun , Park Ho‐Young , Lee Kwang‐Won
Published: June 2021

Many people are interested in well‐being in their lives. Chronic diseases like cancer or hypertension cause low quality of life in those having those diseases. As bioactive substances, fucoidan is helpful to people. Also fucoidan is a polysaccharide with sulfate group and investigated in this study. Fucoidan is well known to be effective in improving the immune system, anti‐virus effect, cardiovascular system, and anti‐cancer effect. The object of this study is to investigate the anti‐tumor effects of fucoidan on lung metastasis in BALB/c mice. Experimental animals were randomly divided into three groups: (1) control group, (2) cancer control group, and (3) group fed fucoidan (60 mg/kg B.W.). Each group was fed orally once per day for 6 weeks. There were no differences of body weights, chow and water intake among three groups. Natural killer (NK) cell activity tested by LDH assay was significantly different (p<0.05) between cancer control group and group fed fucoidan. But, the number of tumor colony and the concentration of NO in serum were not significantantly different among three groups (a control group, a cancer control group, and a group fed fucoidan). We confirmed that fucoidan can improve NK cell activity in lung metastatis in vivo model.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PAX5‐Induced Expression of Endogenous Mucolipin‐2 (MCOLN2) Gene in Human Glial and Neuronal Cell Lines: A Potential Gene Complementation Therapy Approach for Mucolipidosis IV
Authors: Rosas Lauren , Cuajungco Math P.
Published: June 2021

The loss of function in the Transient Receptor Potential Mucolipin‐1 (TRPML1) protein, encoded by the Mucolipin‐1 (MCOLN1) gene, is known to cause the neurodegenerative disease Mucolipidosis type IV (MLIV). Characteristics of MLIV are motor problems, cognitive dysfunction, achlorhydria, cataracts, and blindness caused by retinal cell death. The Ashkenazi Jew population is most afflicted by the disease, and MLIV has no known cure. The Mucolipin ion channels consist of TRPML1, ‐2, and ‐3 proteins, which are encoded by MCOLN1, ‐2, and ‐3 genes, respectively. They are known to serve as non‐selective cation channels located within endosomes and lysosomes and play a role in endosome‐lysosome fusion. Previous research indicates that TRPML1 and TRPML2 proteins share a high degree of homology, and we hypothesize that TRPML2, which has no known association to clinical disease, could substitute for the loss of functional TRPML1 in MLIV, resulting in a rescue of the disease phenotype. In order to study the potential therapeutic effects of TRPML2 in MLIV, we must first show that it is expressed in the brain, one of the tissues most affected by MLIV. We have previously discovered PAX5 as the transcriptional activator for MCOLN2 and have shown that MCOLN2 expression could be heterologously induced in both human neuroglioma (H4) cells and human embryonic kidney 293 (HEK‐293) cell lines. Previous reports demonstrated that MCOLN2 has tissue‐specific expression with little to no detectable transcripts in brain tissue. However, our preliminary research has detected endogenous levels of MCOLN2 transcripts in human H4 and neuroblastoma (SH‐SY5Y) cells. Moreover, to induce the expression of endogenous MCOLN2, we transduced SH‐SY5Y cells with adenovirus and lentivirus vectors containing the PAX5 cDNA. PAX5 serves as a B cell‐specific activator protein with tissue‐specific expression pattern as well. Standard reverse‐transcription polymerase chain reaction (RT‐PCR) and real‐time quantitative RT‐PCR both revealed MCOLN2 expression in SH‐SY5Y cells at 48 and 72 hours post‐transduction. We show that PAX5 transcriptionally activates and increases endogenous MCOLN2 expression in neuronal cells that are central to studying MLIV. Future studies include In‐Cell Western assay with an anti‐TRPML2 antibody and transcriptome analysis of PAX5‐treated cells to determine global gene expression. These results and future studies will further explore the therapeutic potential of gene complementation to rescue the cellular phenotype in MLIV disease.

Support or Funding Information

This work was partly funded by NIH R15 NS101594.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Use of the Phytochemical Neem as a Component of Combination Treatment Against Pseudomonas aeruginosa Biofilm
Authors: Bertrand Blake Patrick , Heuertz Rita M.
Published: June 2021

According to the NIH, 80% of chronic infections are caused by biofilm‐associated bacteria. Biofilm communities contribute to antimicrobic resistance by providing a physical extracellular barrier and exhibit metabolic diversity within bacterial subpopulations. Clinically, combination antibiotic strategies are used to provide high dosage treatments to successfully combat biofilm‐associated infections. Microscopically, biofilms of Pseudomonas aeruginosa have been visualized as mushroom‐like structures with two distinct subpopulations: a high‐metabolic “mushroom cap” outer population and a low‐metabolic “stalk” inner population. Further, the inner subpopulation consists of persister cells that are not killed by many therapies and contribute to chronicity of biofilm‐associated infections. It has been proposed that complete eradication of biofilm is achieved by targeting specific biofilm populations with antimicrobic combination of colistin and ciprofloxacin that target inner and outer subpopulations of biofilm respectively. However, colistin is sparingly used clinically because of high renal toxicity. Use of phytochemical alternatives instead of antibiotics is under assessment for treatment of biofilm‐associated infections. Extract from Azadirachta indica, or neem tree, is reported to have antimicrobic and antibiofilm effects on P. aeruginosa with low human toxicity. For these reasons, P. aeruginosa was grown (24 h) and microplates washed to remove non‐adherent cells. Remaining biofilm and cells were treated overnight with neem, colistin, ciprofloxacin or dual combinations. Finally, biofilm was removed from the substratum by scraping and aspiration, and bacterial viability was determined by colony forming unit (CFU/mL) enumeration. Results indicated that neem and ciprofloxacin in combination had an inhibitory effect (≤103 CFU/ml) similar to colistin and ciprofloxacin combination (≤102 CFU/ml) compared to growth and vehicle controls (both of which were 1012 CFU/ml). Colistin and neem combination showed no combined effect (107 CFU/ml) different from single treatment effects (108 and 1010 CFU/ml, respectively). These results suggest that neem extract targets the inner, low‐metabolic subpopulation of bacteria in biofilms and, for this reason, neem has potential to be a non‐toxic alternative to colistin for use therapeutically. When colistin or neem (that target the inner, low‐metabolic bacterial subpopulation) are used in combination with ciprofloxacin (that targets the outer, high‐metabolic subpopulation), the entirety of the biofilm may be targeted and prevent chronicity in biofilm‐associated infections.

Support or Funding Information

Financial support for BPB was received from the DeNardo Education and Research 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.
Subcellular Localization of The Enzymes In Serine Biosynthesis
Authors: Nance Bailey , Kyoung Minjoung , An Songon
Published: June 2021

Serine biosynthesis uses 3‐phosphoglycerate, an intermediate of glycolysis, to catalyze the first reaction by phosphoglycerate dehydrogenase (PHGDH). This reaction produces 3‐phosphohydroxypyruvate, which eventually becomes serine through two additional steps. Upregulation of serine biosynthesis has been characterized as a hallmark of cancer cell metabolism, as serine is an essential metabolite that provides precursors for various building blocks supporting cancer cell growth and proliferation. We hypothesize that the enzymes involved in serine biosynthesis may associate with each other in the cytoplasm of human cancer cells. To test this hypothesis, we use molecular cloning techniques to tag human PHGDH and others with a fluorescent protein. We will then transfect the plasmids into human cancer cells to examine the localization of PHGDH and others using fluorescent live‐cell imaging. Ultimately, we will contribute to our understanding of the spatiotemporal regulation of serine biosynthesis in human cancer cells.

Support or Funding Information

This work was supported by UMBC Start‐up fund to S.A. This investigation was also sponsored in part by NIH/NIGMS MARC U*STAR T34 08663 National Research Service Award to B.N.

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