bluuebunny/crossref_metadata_2025 huggingface dataset:
Utilizing Design Principles of Electrochemical DNA Aptamer Biosensors in Multiple Optical Assay Methodologies
Authors: Bonham Andrew J.
Published: June 2021

DNA aptamers, short oligonucleotides with high specificity and affinity for a variety of molecular targets, have been widely adopted as capture probes and detection elements for a variety of assay and biosensor applications. In particular, electrochemical DNA (E‐DNA) biosensors have been demonstrated for many applications, ranging from the detection of metal ions (e.g., uranium and mercury), to small molecules (e.g., doxorubicin and other antibiotics), and even to large proteins (e.g., botulinum toxin and transcription factors). E‐DNA biosensors essentially consist of an aptamer oligonucleotide region that is incorporated into a larger oligonucleotide scaffold, with an anchoring modification (typically a thiol) and a reporter modification (typically the dye and redox reporter methylene blue) covalently appended. Recent efforts in our lab have focused on taking insights from E‐DNA biosensor design and incorporation of aptamers into functional probes, and applying them to a variety of alternative, optical assays repurposing existing E‐DNA probes. One of these approaches is the demonstration of Raman spectroscopic detection through dye‐labeled DNA functionalized gold nanoparticle assemblies that dissociate in a dose‐dependent response to the presence of the target molecule. A second approach has been the creation of portable, do‐it‐yourself 3D‐printed total internal reflectance fluorescence based assays using dye‐labeled DNA arrays on acrylate microscope slides where fluorescence intensity, which can be captured on a smart phone, reflects target presence. A third approach has focused on the characterization of rapid lateral flow assay (“dipstick”) assays based on DNA functionalized gold nanoparticles and nitrocellulose arrays, where target presence is reflected in the intensity of a nanoparticle‐based color line. Ultimately, these projects illustrate the high transferability of DNA aptamer biosensor design parameters between different sensing modalities, offering insight into engineering successful biosensor designs.

Support or Funding Information

This work is supported by internal grant funding from the Metropolitan State University of Denver.

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 novel suite of genetically encoded fluorescent biosensors for dynamic and sensitive enzyme activity measurements in vivo
Authors: Mehta Sohum , Mo Albert , Zhang Jinfan , Zhang Jin
Published: June 2021

Fluorescent protein‐based biosensors have revolutionized biomedical science by enabling the direct, real‐time visualization of molecular processes with high spatial and temporal resolution in living cells. In particular, biosensors based on fluorescence resonance energy transfer (FRET) are among the most versatile and widely used probes for investigating the dynamic, spatiotemporal regulation of enzymatic activities in the native biological context. Yet the transformative potential of these genetically encoded molecular tools is constrained by their generally low dynamic ranges, which limits their sensitivity in more physiologically and clinically relevant settings (i.e., in vivo). We have developed and optimized a novel class of genetically encoded enzyme activity biosensors whose dynamic ranges exceed those of even the best‐performing FRET‐based activity reporters by several decades and are easily the highest ever reported for genetically encoded enzyme activity reporters. These high‐performance molecular tools permit the robust, sensitive detection of subtle activity changes in vivo and thus further expand the frontiers of “native biochemistry”.

Support or Funding Information

This work was supported by Brain Initiative Grant R01 MH111516 and by R35 CA197622, R01 DK073368, and R01 GM111665 (to J.Z.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lipidomics reveals physiological isotope effects during the enzymatic oxygenation of polyunsaturated fatty acids ex vivo.
Authors: Navratil Aaron R. , Shchepinov Mikhail S. , Dennis Edward A.
Published: June 2021

Arachidonic acid (AA, 20:4) is an omega‐6 polyunsaturated fatty acid (PUFA) and the main precursor to the class of lipid mediators known as eicosanoids. The enzymes that catalyze the oxygenation of AA begin by abstracting hydrogen from one of three bis‐allylic carbons within 1,4‐cis, cis‐diene units. Replacing bis‐allylic hydrogen atoms with deuterium has been shown to strongly slow the rate of enzymatic oxygenation of linoleic acid by soybean lipoxygenase, resulting in a kinetic isotope effect (KIE). Yet, experimental determination of the KIE during oxygenation of AA by mammalian cyclooxygenase (COX) and lipoxygenase (LOX) has revealed far lower values. All prior studies investigating the KIE of PUFA oxygenation have relied on in vitro systems using purified enzymes and were limited by availability of deuterated PUFAs. Here we demonstrate the use of macrophages as an ex vivo model system to study the physiological kinetic isotope effect (PKIE) during enzymatic AA oxygenation by living cells using a newly synthesized library of deuterated AA isotopologues. By extending sensitive lipidomic UPLC‐MS/MS approaches to simultaneously quantify native and deuterated lipid products, we were able to measure the magnitude of the PKIE for COX and LOX oxygenation of AA by macrophages. This work provides the first demonstration that hydrogen(s) present at carbon 10 (C10) of AA play an important role in the catalysis of prostaglandin and thromboxane synthesis. Furthermore, we discovered that deuteration of C10 also promotes the formation of the resolving lipid mediator lipoxin B4 by activated macrophages, possibly by interfering with AA cyclization and shunting AA toward LOX metabolism under physiological conditions.

Support or Funding Information

NIH RO1 GM20501‐41

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metabolic Markers of Hyperhomocysteinemia in Healthy Adults Population
Authors: Waly Mostafa I. , Ali Amanat , Padmanabhan Smitha , Al‐Farsi Yahya
Published: June 2021

Background

Hyperhomocysteinemia is involved in the pathogenesis of oxidative stressmediated human chronic diseases including; cancer, cardiovascular diseases, hypertension, and type 2 diabetes. B vitamins (folate, vitamin B6, and vitamin B12) deficiencies induce hyperhomocysteinemia, and thereby contributes to the pathology of human chronic diseases.

Objective

This study aimed to assess the biochemical and nutritional status of B vitamins in relation to hyperhomocysteinemia among healthy adults population.

Methods

The study participants (n=400; 190 males and 210 females) were healthy adults subjects and free of any chronic diseases or endocrine disorders. They were supplied with B vitamins for 6 months and the homocysteine and B vitamins levels were measured twice in the sera of the study subjects (before starting the trial and at the end of the trial). The nutritional status was assessed using prospective semi‐quantitative food frequency questionnaire.

Results

Low B vitamins levels were observed in the sera of study participants with hyperhomocysteinemia (fasting serum homocysteine >15 μmol/l). Meanwhile, B vitamins supplementation was effective in combating hyperhomocysteinemia and its associated oxidative stress indices as evidenced by enzymatic and non‐enzymatic antioxidants markers.

Conclusion

Taking adults population with hyperhomocysteinemia as a high risk group; the results of this study indicate that B vitamins supplementation modulates hyperhomocysteinemia‐mediated oxidative stress. Serum B vitamins levels might be used as a novel biomarker for early diagnosis and screening of hyperhomocysteinemia,

Support or Funding Information

This research was supported by Internal Grant Funding (IG/AGR/FOOD/17/1), College of Agricultural and Marine Sciences, Sultan Qaboos University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Role of FLO1 in the Lack of Flocculation of Yeast Brewing Strain Thirsty Pagan Local
Authors: Rieffer Amanda Elizabeth , Via Zachary
Published: June 2021

Brewer's yeast use a fermentation pathway to convert sugars into ethanol in the beer making process. An interesting characteristic most yeast have is the ability to clump together, known as flocculation. Flocculation is useful for brewing companies because it makes it much simpler to separate and reuse it for later brews. Our research is focused on a locally found strain of Saccharomyces cerevisiae, Thirsty Pagan Local (TPL), that does not flocculate, and we want to know why. There are a series of genes associated with flocculation, and FLO1 codes for a protein on the surface of the cell that aids in flocculation. An ethanol sensitivity assay was conducted, and based on this know that FLO1 for TPL is not a null mutant, so it's possible that this gene is simply under expressed. When comparing TPL FLO1 with wild type DL1 FLO1 (a known nonflocculator), we noticed that our FLO1 is roughly 1,000 base pairs shorter than wild type. We are in the process of sequencing TPL FLO1 to find areas where there is a detrimental amino acid change that could give us a clue as to why our strain doesn't flocculate. We have successfully inserted FLO1 into an overexpression plasmid, and plan on conducting a flocculation assay with this plasmid along with FLO1 from a known flocculator, 1195. If the overexpression of TPL FLO1 results in flocculation, our next plan is to establish the overexpression vector permanently into the yeast genome.

Support or Funding Information

The College of St. Scholastica

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 Impact of the Physical Micro‐Environment on Notch Signaling
Authors: Detweiler Michael Allen
Published: June 2021

Notch is a transmembrane ligand receptor that is involved in cell‐cell communication via canonical binding to a mounted ligand on an adjacent cell. Upon activation of ligand binding, Notch's intracellular portion is cleaved and translocates to the nucleus to function as a transcriptional regulator of many downstream genes. Extracellular micro‐environmental cues including shear stress, ECM stiffness, pH, etc. have been shown to impact Notch activation. Integrins are dimerizing proteins that aid in attachment of the cell to the extracellular matrix, which can have impacts on cell physiology from the micro‐environment. Recently published data has also revealed that integrins have an effect on Notch activation, but the mechanism(s) inducing this response have yet to be discovered. Because the extracellular micro‐environments can impact cell physiology through integrin functions, we hypothesize that these physical micro‐environmental conditions may also impact Notch signaling. To investigate this hypothesis, we monitored Notch activation during shear stress or increasing extracellular matrix stiffness, two conditions shown to influence integrin downstream signaling. Our preliminary results show that applying shear stress on endothelial cells provides an increase in downstream Notch signaling activity, which is effected by inhibiting integrin‐linked molecules. Our preliminary results also suggest that Notch activity is controlled by matrix stiffness and that inhibiting Notch activation on stiff extracellular matrix impacts the production of smooth muscle actin (indicator of fibrosis), suggesting a pathway to fibrosis through an integrin‐Notch connection. Together, these experiments support our hypothesis of the impact of the physical microenvironment on Notch signaling through integrin activation. By coming to these conclusions, we hope to gain a better understanding of the impacts integrins and the microenvironment have on Notch signaling and to identify the mechanisms involved.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparison Between the Structure‐Function Relationships in Oncogenic and Wild‐Type Gα Subunits
Authors: Goossens Jesse , Leverson Brian , Freitas Duarte
Published: June 2021

Guanine nucleotide binding proteins (G‐proteins), in conjunction with their receptors (GPCR), are one of the most prevalent signaling systems and are involved in the regulation of an array of cellular processes. Upon binding of an extracellular stimulus to the GPCR, the activated α‐subunit (Gα) exchanges GDP for GTP, releases both the βγ‐complex and the GPCR, and associates with an effector that further relays the signal. The signal terminates with the hydrolysis of GTP to GDP, causing the α‐subunit to reunite with the βγ‐subunits and the GPCR. G‐protein signaling must be tightly regulated to ensure the appropriate responses to extracellular stimuli. Any misregulation caused by mutations can be detrimental. Gsα and Giα1 are G‐protein subunits that, respectively, stimulate or inhibit adenylyl cyclase and regulate the production of the secondary messenger, cAMP. We studied two mutations observed in cancers of the intestine (R231H mutation in Gsα) and the colon (R208Q mutation in Giα1). Spectroscopic and crystallographic approaches were used to further understand these disease states.

Fluorescence spectroscopy, circular dichroism, and UV/Vis spectroscopy showed lower melting temperature in the active conformation of the oncogenic mutants, indicating disruptions in the noncovalent interactions. Using malachite green and fluorescence GTP assays, we found that the kcat values for oncogenic Giα1 were lower than for the WT protein; the trend was the opposite for the WT and oncogenic Gsα proteins. When comparing the x‐ray structure of the R208Q mutant with that of the WT Giα1, the oncogenic point mutations do not show significant differences in the structure of the protein as a whole. However, molecular dynamics simulations modeling the interactions of T181 in Giα1 and T204 in Gsα (key residues in the hydrolysis of GTP) show drastically different interaction energies between mutants and WT proteins. Taken together, these findings suggest that the altered rates of GTP hydrolysis lead to an imbalance of cAMP in tumor cells.

Support or Funding Information

NIH R15GM112025

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidating structure‐function relationships of the human protein kinase MEK1
Authors: Lee Rebecca T. , Ravatt Leandre M. , Runco Caroline E. , Oza Javin P.
Published: June 2021

MEK1 is a dual specificity protein kinase in the MAPK phosphorylation cascade. As one of the prevalent signal transduction pathways, the MAPK pathway regulates a diverse variety of cellular processes including proliferation, differentiation, and apoptosis. The phosphorylation event between MEK1 and its only known substrate ERK2 acts as regulatory bottleneck for signal transduction, and maintains the fidelity of signals coming in and going out. As such, MEK1 remains a target for therapeutic intervention of a variety of cancers. While much is known about MEK1's role in cell signaling, enzymatic characterization of MEK1 has been limited due to difficulty in obtaining the active, doubly‐phosphorylated MEK1 in a homogenous population. Our study overcomes this barrier by utilizing an expanded genetic code for the co‐translational incorporation of phosphoserine into MEK1. Specifically, we are leveraging this capacity for on‐demand production of site‐specifically phosphorylated MEK1 to characterize the human MEK1 and elucidate the structure‐function relationships that governs MEK1 activity. Using a hypothesis‐driven approach based on conserved kinase motifs, we selected residues within the activation segment and catalytic loop for site‐specific mutagenesis. Here we report our efforts to express and characterize the phosphorylated MEK1 and its mutants through immunoassays for a comparative analysis of kinase activity. Our work aims to establish kinetic parameters for the human MEK1 for the first time and provide insights into mechanisms of substrate binding and catalysis.

Support or Funding Information

Our research is funded by the Center for Applications in Biotechnology @ Cal Poly and the Bill and Linda Frost Fund.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein Kinase Cα (PKCα) Gain‐of‐Function Variant in Alzheimer's Disease Displays Enhanced Catalysis by a Mechanism that Evades Down‐Regulation
Authors: Callender Julia , Yang Yimin , Stephenson Natalie , Jones Alexander , Brognard John , Newton Alexandra
Published: June 2021

The signaling output of protein kinase C (PKC) is precisely controlled through multiple mechanisms that balance its activity in order to maintain cellular homeostasis, with disruption of this balance resulting in pathophysiologies. In this vein, the cell has evolved mechanisms to maintain this balance by degrading constitutively active PKCα. We have previously shown that loss‐of‐function mutations in multiple PKC isozymes contribute to cancer, a disease of aberrant proliferation, and gain‐of‐function mutations in the conventional isozyme PKCα contribute to Alzheimer's Disease (AD), a disease of aberrant cell death. Here, we biochemically characterize one of the AD‐associated variants, PKCα‐M489V, in order to investigate the mechanism through which it confers enhanced signaling output without causing PKCα degradation. We use a combination of in vitro and in silico methods to demonstrate that this mutation effectively increases the specific activity of purified PKCα under activating conditions by increasing the molecular dynamics of the catalytic domain. Importantly, it does so without affecting the normal autoinhibition of basal signaling, thereby allowing for an enhanced signaling output that simultaneously evades the cell's homeostatic degradation of PKCα protein levels. Taken together, our results provide an in‐depth characterization of a disease‐associated gain‐of‐function PKC mutation, and for the first time report a mutation that enhances signaling output of PKCα while maintaining its stability in the cell.

Support or Funding Information

This work was supported by NIH R35 GM122523 and NIH GM43154 (ACN), the Cure Alzheimer's Fund (ACN), and Cancer Research UK (NS, JB). JC was supported by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology (T32 GM007752).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhancing Scientific Communication About and Ethical Reasoning Surrounding Gene Modifications Through a Collaborative Learning Experience
Authors: Danowitz Amy M. , Ehret Verna M.
Published: June 2021

Scientific communication and the ability to think about the ethical impact if one's work are necessary skills for all scientists to obtain. These are especially important skills for scientists working in cutting edge fields that have the potential for major societal implications, including scientists working with genetic modifications of humans and animals. Unfortunately, the traditional undergraduate curriculum generally does not provide many opportunities for students to practice communicating their science to the general public, nor does it offer much ethical training beyond standard responsible conduct of research requirements. Herein, we describe a novel collaborative learning experience that allows students who are studying modern genetic modification techniques (including traditional methods of gene modification, as well as newer advances such as RNAi and CRISPR/cas9) to work with students who are studying ethical reasoning. As part of this collaboration, the science students are required to hone their understanding of a technique used in genetic modification and to explain this technique to a largely non‐scientific audience (the ethics students). The ethics students, in turn, teach the science students about some of the ethical concerns that can arise around these techniques. By participating in this experience, students gain a better understanding of how to best communicate complex, technical information with non‐scientists and a fuller idea of how ethical decision making can impact their lives as laboratory scientists and as citizens of the world. Both the faculty and the students involved in this collaboration found this to be a worthwhile and enjoyable experience. The faculty were particularly impressed by the high levels of student work and thought put into this project. This poster will address the assignments that were given, the grading standards for these assignments, general student opinions on this project, and ideas for where and when to fit such collaborative projects into the undergraduate curriculum.

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 Toxic Collaboration: Using SPIONS and Shrimp to Bridge Courses in Chemistry and Biology for Undergraduate Engineering Students.
Authors: Rogers Ryan Patricia , Grove Laurie Eileen
Published: June 2021

Chemistry and biology are naturally symbiotic disciplines, which provide foundation for other related areas such as engineering. At Wentworth Institute of Technology, the majority of students enroll in our Engineering Chemistry course and many others will also take Cellular and Molecular Biology, either to fulfill a major requirement or science elective. It is between these two courses that we have designed a collaborative project investigating the synthesis and biological relevance of superparamagnetic iron oxide nanoparticles (SPIONs). SPIONs are at the forefront of investigation across several different fields, which makes them an exceptional interdisciplinary educational tool. This project specifically focuses on the role of SPIONs as contrast agents for MRI, which directly relates to future coursework for our biomedical and biological engineering majors; however, the construction of the NMR and MRI instruments, including the superconducting coil, provides interest for our electrical and mechanical engineering students. Students in chemistry are paired with cohorts of biology students and directed to compile their experimental data to generate a complete narrative related to the synthesis and uptake of SPIONs by brine shrimp. Numerous synthetic variations of SPIONs are available, thus allowing students to develop and test a hypothesis relating to either the magnetic properties or toxicity of the nanoparticles, which can only be explained and supported at the conclusion of both experiments.

The collaboration culminates with a video lab report, developed and presented by both cohorts of students. Scores are generated based on presentation style and creativity, scientific accuracy, ability to develop and explain their hypothesis, data analysis and interpretation of findings on a larger scale. The collaboration takes place over the course of 3 2‐hour lab periods, with additional check points during the biology component. Two of these periods are designated for synthesis and characterization of SPIONs by the chemistry students and one lab period is designated for induction of toxicity assays and cytotoxicity analysis by the biology students.

Support or Funding Information

This work was supported by the Department of Sciences at Wentworth Institute of Technology.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Paclitaxel on Glucocorticoid‐induced apoptosis of Human Leukemic CEM Cells
Authors: Aranas Ivymarie , Medh Rheem
Published: June 2021

Glucocorticoids (GCs) are steroid hormones widely used in anti‐leukemia therapy as they can induce apoptosis through certain gene‐regulatory pathways that affect pro‐ and anti‐apoptotic genes. In cell culture models of human acute lymphoblastic leukemia (ALL), the synthetic GC, Dexamethasone (Dex), has been shown to induce expression of pro‐apoptotic genes including E4BP4 (a transcription factor that upregulates pro‐apoptotic BIM), and regulators of apoptosis such as FOXO3 and BIRC3, in correlation with apoptosis. Paclitaxel (Ptx), a taxol derived anti‐proliferative alkaloid, induces apoptosis via a distinct but overlapping transcriptional response, and is often used in combination with Dex as an anti‐leukemic agent. In this study we are investigating the effect of Ptx alone, and in combination with Dex, on apoptosis and regulation of key pro‐ and anti‐apoptotic genes, in an effort to understand the molecular implications of combined treatment with Dex and Ptx. The CCRF‐CEM cell lines CEM‐C7‐14 and CEM‐C1‐15 were previously found to be sensitive and resistant, respectively, to Dex. Sensitivity to Dex was restored in CEM‐C1‐15 cells upon ectopic expression of mouse E4BP4 (line CEM‐C1‐15mE#3). We are testing the hypothesis that E4BP4 and BIM play a central role in regulating apoptosis triggered by Ptx as well, and that combined treatment synergistically triggers apoptosis via a greater magnitude of upregulation, in addition to activating a broader spectrum of pro‐apoptotic pathways. All three CEM cell lines were treated with 1 mM Dex and 1mM Ptx either alone or in combination for 24–96h. All three cell lines were sensitive to Ptx, but CEM‐C1‐15mE#3 was least susceptible. Apoptosis was confirmed by staining cells with Annexin V‐FITC followed by epifluorescence microscopy. Reverse transcription and end‐point PCR followed by ImageJ analysis was employed to evaluate relative gene expression in response to each treatment. Our data suggest that Ptx upregulated FOXO3, BIRC3, and BIM, but down‐regulated E4BP4 in the CEM‐C7‐14 cells, suggesting that Dex and Ptx have distinct effects on E4BP4 regulation but may share common downstream pathways for apoptosis via BIM upregulation.

Support or Funding Information

NIH SC3 grant, #GM081099, and a Building Infrastructure Leading to Diversity (BUILD) grant from the NIH, #5RL5MD009603

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Primary Acute Lymphoblastic Leukemia Cells Strictly Require Interphase Microtubules for Survival during G1 Phase Advance
Authors: Delgado Magdalena , Chambers Timothy C.
Published: June 2021

Microtubule targeting agents (MTAs), such as vincristine (VCR), are important anticancer agents and are thought to act primarily in mitosis. However, there is increasing evidence that MTAs can also cause death by interfering with interphase microtubules. We have recently discovered that when primary acute lymphoblastic leukemia (ALL) cells are treated with vincristine, cells in G1 phase die directly without advancing to other phases of the cell cycle. This system represents a powerful model for understanding interphase mechanisms of MTA action, and provides an opportunity to test whether MTAs can be combined with agents that target interphase processes in order to create novel treatments for ALL. One such candidate is palbociclib (PCB), a highly selective inhibitor of cyclin dependent kinases 4/6 (CDK4/6), whose activity is necessary for the transition from G1 to S phase, providing a rationale for the combination of palbociclib and vincristine to target ALL cells in G1 phase. In this study, we tested the effect of PCB alone or in combination with vincristine in primary ALL cells. PCB caused arrest of primary ALL cells in G1 phase, with 97–99% of cells exhibiting 2N DNA content, while having no significant effect on cell viability. In contrast, HeLa cells were unaffected by PCB, consistent with their lack of dependence on the CDK4/6‐retinoblastoma pathway. When ALL cells were pretreated with PCB, they became refractory to VCR treatment. Examination of the microtubule network by immunofluorescence microscopy in ALL cells treated with PCB alone or PCB followed by VCR treatment showed that PCB did not disrupt the microtubule network, nor prevented VCR from doing so. Furthermore, ALL cells treated with ABT‐263, a Bcl‐2/Bcl‐xl inhibitor, after PCB pretreatment still underwent cell death, indicating that PCB did not block downstream apoptotic pathways. Experiments assessing entry and passage through S phase using 5‐ethynyl‐2′‐deoxyuridine incorporation showed that G1 arrest in primary ALL cells by PCB was reversible, and that cells released from arrest regained susceptibility to VCR. Taken together, these results indicate that ALL cells must be cycling in order for interphase death induced by vincristine to occur, and in turn indicate that interphase microtubules play a fundamental role in survival of actively cycling ALL cells. In addition, these findings strongly caution against combining vincristine and CDK4/6 inhibition as a potential therapy for ALL.

Support or Funding Information

Chancellor's Circle Award

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hepatotoxicity of Ochratoxin A, Benzo [a] pyrene and Acrylamide, Alone and in Combination with HepG2 Cell through Phase I and Phase II Pathway
Authors: Shin Hye Soo , Pyo Min Cheol , Bae Hyun‐Sun , Bae Jung Min , Lee Kwang‐Won
Published: June 2021

Ochratoxin A (OTA) is a widespread mycotoxin produced by several species of the genera Aspergillus and Penicillium. Since OTA is mainly found in foods such as rice and oats consumed all over the world, there are many researches on the reduction of OTA. Benzo [a] pyrene (B[a]P) and acrylamide (AA) may occur during the heat treatment process to reduce the OTA present in rice. In this study, we tried to identify the combined toxicity of the three substances (OTA, B[a]P, AA) in the liver. The cell viability of OTA, B[a]P and AA was checked to determine the concentration of IC20. The toxicity of the combination of the three substances showed synergistic effects on cell viability and NO production. To further elucidate the mechanism of toxicity in liver, real‐time PCR was used to confirm mRNA expression levels of enzymes involved in phase I and II pathway. In the case of cytochrome P450 (CYP) families (CYP1A1, CYP1A2, and CYP3A4) belonging to Phase I, the mRNA expression levels were significantly increased when all three of them were mixed with each other than those treated with OTA, B[a]P and AA, respectively. The mRNA expression levels of heme oxygenase‐1 (HO‐1) and glutamate cysteine ligase catalytic subunit (GCLC) belonging to Phase II were also similar to those of Phase I. Therefore, in all experiments, the combined toxicity of OTA, B[a]P, and AA was confirmed, indicating synergistic effects on hepatotoxicity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Coordinated Cross‐talk between calcium and cAMP in regulating pulsatile insulin secretion: A novel role for the unique inhibitory G‐protein, Gαz, in regulating β‐cell function
Authors: Schaid Michael , Harington Jeff , Wienkes Haley , Merrins Mathew , Kimple Michelle
Published: June 2021

Cyclic adenosine monophosphate (cAMP) is an important secondary messenger involved in a variety of signal transduction mechanisms. In the pancreatic β‐cell, cAMP amplifies pulsatile insulin secretion through protein kinase A (PKA) and the exchange protein activated by cAMP (EPAC). Inhibition of adenylyl cyclase (AC) limits glucose stimulated insulin secretion, even in the absence of activated stimulatory proteins, suggesting an essential role of cAMP in β‐cell function. It has been proposed that cAMP may coordinate with calcium oscillations to regulate pulsatile insulin secretion; however, this has not been demonstrated in intact islets under physiological conditions. In this study we utilize a β‐cell‐specific, genetically encoded cAMP biosensor expressed in intact isolated mouse islets to demonstrate for the first time an anti‐phasic relationship between cAMP and Ca2+ oscillations. This suggests that intracellular cAMP levels are governed by the Ca2+ sensitive phosphodiesterase, which act to degrade intracellular cAMP during the active phase of insulin exocytosis. Furthermore, loss of the inhibitory G‐protein alpha‐subunit, Gαz, an important contributor to diabetes pathology, increased the amplitude of cAMP oscillations and sensitivity to glucagon like peptide‐1 (GLP‐1) receptor stimulation. Ligand activation of E‐prostanoid receptor 3 (EP3), a G‐protein coupled receptor (GPCR) up regulated in the diabetic state that couples to Gαz, reduces cAMP oscillations but also dramatically blunts Ca2+ oscillations. These results suggest a coordinated cross‐talk between Ca2+ and cAMP dynamics in the β‐cell, as well as a novel role for Gz signaling which acts on both Ca2+ dynamics and inhibition of the cAMP‐mediated amplifying pathway of insulin secretion.

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 BIOLOGICAL IMPACT OF THE GOLGI MEMBRANE PROTEIN TMEM165 FOR BREAST CANCER
Authors: Murali Pavitra , Johnson Blake , Climer Leslie , Oprea‐Ilies Gabriela , Lupashin Vladimir , Abbott Karen
Published: June 2021

The TMEM165 gene encodes for a multiple pass membrane protein, localized in the Golgi that has been linked to congenital disorders of glycosylation. We have identified TMEM165 as a biomarker for breast carcinoma in a previous glycoproteomic study. The TMEM165 protein is not expressed in non‐malignant breast tissue, increases slightly in early stage ductal carcinoma in situ (DCIS) cases, and is highly expressed in invasive ductal breast cancer. Our hypothesis is that TMEM165 confers a growth advantage to breast cancer. In this preliminary study we created a CRISPR/Cas9 knockout of TMEM165 in the human invasive breast cancer cell line MDAMB231. Our results indicate that removal of TMEM165 in these cells results in a significant reduction of cell migration and tumor growth in vivo. These studies illustrate new potential functions for this Golgi membrane protein in the control of tumor cell growth and invasion.

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 PKHD1L1's Localization and Potential Function as a Tumor Suppressor
Authors: Shi Xingyi , Yoo Lina
Published: June 2021

The Polycystic Kidney and Hepatic Disease 1‐like 1 (PKHD1L1) gene located on human chromosome 8 has been shown to have a high frequency of mutation in multiple types of epithelial cancers, such as skin, lung, and bladder cancer, according to data generated by the TCGA Research Network; its mutation rate in bladder urothelial carcinoma (11.41%) was ranked 65 among 19,578 genes analyzed. Furthermore, the TCGA data demonstrated that PKHD1L1 gene expression was diminished compared to normal tissues in those epithelial tumors. Our approach was to examine PKHD1L1's potential tumor suppression functions in bladder cancer cell line UM‐UC‐3. We engineered a CRISPR‐Cas9 PKHD1L1‐gRNA expression vector, which was then transfected into UM‐UC‐3 cells. After selection and establishment of clonal cell lines, mutation of full‐length PKHD1L1 was confirmed to be successful via TIDE analysis showing complete, frame‐shift disruptions. We performed wound healing assays, MTS assays, and colony formation assays to examine the impact of full‐length PKHD1L1 deficiency on cell motility and proliferative capacity. Wound healing assays and MTS assays showed no significant difference due to PKHD1L1 deficiency. Preliminary studies suggest PKHD1L1 deficiency may result in larger and more numerous colonies in the colony formation assay. We are currently investigating the subcellular localization of PKHD1L1 protein through immunocytochemistry. With further investigation, our research can provide significant insight into the role of PKHD1L1 in cancer development and progression.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Correlation of Oyster Disease with Vibrio Parahaemolyticus accumulation in oyster tissue
Authors: Jackson Kiserian , Scro Abigail , Smolowitz Roxanna
Published: June 2021

As the average water temperature of Narragansett Bay and other coastal ponds in Rhode Island continue to increase due to climate change it is probable that the abundance of Vibrio parahaemolyticus (Vp) and Vibrio vulnificus (Vv) will increase as well. Vibrio are gram‐negative bacteria that can accumulate in tissues of the eastern oyster (Crasssostrea virginica). While vibrio is not pathogenic to the oyster it can cause gastroenteritis in humans that consume the raw oysters. Eastern oysters are commonly infected with three diseases: Perkinsus marinus (Dermo), Haplosporidium nelson (MSX) and Haplosporidium costale (SSO). These parasites can cause severe morbidity and mortality in cultured and wild oysters. Animals that are affected by any disease have a decreased immune functionality and it is probable that these diseased oysters are more likely to accumulate higher levels of Vp and Vv. To test this theory, oysters were collected from Roger Williams University during the summers of 2016 and 2017. Composites of 10 oysters were tested using multiplex qPCRs for Vp and Vv in conjunction with a second multiplex qPCR to determine the abundance of those three disease. Individuals were also tested in order to increase accuracy of the association because composites which produce averages may mask important relationships only identified in individual animals.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dendropanax improve kidney function by inhibiting oxidative stress via upregulating sirt‐1 in Streptozotocin‐induced diabetic rats
Authors: Sachan Richa
Published: June 2021

Objective

The objective of the study was to evaluate the effect of Dendropanax (DP) for its efficiency in ameliorating diabetes and its associated nephropathy with its possible underlying protective mechanisms.

The present study was designed to evaluate the protective effect of Dendropanax (DP) morbifera against nephrotoxicity in streptozotocin (STZ)‐induced diabetic rats. Diabetes was induced by a single injection streptozotocin (50 mg/kg, i.p.). The water extract of DP (400 mg/kg body weight) was administered by oral gavage for 4 week. We measured body weight changes, blood lipid parameters, blood glucose, glucose tolerance, insulin sensitization, blood urea nitrogen (BUN), creatinine, AST, or ALT. In addition, we measured the expression of SIRT‐1, extracellular matrix, oxidative stress, and fibrosis markers. Administration of DP in STZ‐induced diabetic rats significantly improved glucose tolerance and insulin sensitization. The levels of serum glucose, insulin, total protein, albumin, triglycerides, cholesterol, low density lipoprotein cholesterol (LDL‐C), creatinine was significantly increased in STZ‐induced diabetic rats. In diabetic rat model, DP significantly reversed the serum levels of BUN and creatinine and decreased serum 3‐indoxyl sulfate levels, a new nephrotoxicity biomarker in STZ‐induced rats. In particular, the protective effects of DP on diabetic‐induced renal injury were clearly exhibited by analysis of histopathological examination in STZ‐induced rats. In particular, oxidative stress‐mediated pathway in diabetic nephropathy was closely associated with NF‐kB and TGF‐β1 pathways. The protective effects of DP on diabetic‐induced renal injury were clearly exhibited by analysis of histopathological examination on the kidney. We demonstrated that DP extracts protects diabetic‐induced renal injury by blocking oxidative damage via SIRT‐1 activation which indicating its clinical application to inhibit diabetic‐induced chronic kidney disease (CKD).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antihyperglycemic, nephroprotective and antioxidative effects of white and yellow Discorea domentorum yam based‐diet in alloxan‐induced diabetic rats
Authors: Ajayi Olubunmi Bolanle
Published: June 2021

The present study was convened to assess the effects of white and yellow Discorea domentorum yam (Trifoliate yam tubers) based‐diet on biochemical parameters in alloxan‐induced diabetic rats for 14 days. Experimental rats were divided into four groups (n=6): Group 1: normoglycemic group which were fed corn starch‐based diets; group 2: diabetic control group fed corn starch based diet; group 3 and 4 served as diabetic test groups fed white and yellow Discorea domentorum yam flour‐based diets respectively. The results revealed elevation of blood glucose levels in diabetic control rats fed corn starch‐based diets when compared to the normal control rats. Moreover, increased blood glucose levels were suppressed significantly (P < 0.05) in diabetic rats fed white and yellow trifoliate‐based diets. Furthermore, reduced activities of superoxide dismutase (SOD) and catalase (CAT) as well as decreased levels of reduced glutathione (GSH) and malondialdehyde (MDA) were observed in diabetic control rats fed corn starch‐based diets when compared to normal control rats and diabetic rats fed white and yellow Discorea domentorum yam flour‐based diets. In addition, urea and creatinine levels were significantly (P < 0.05) increased in diabetic control rats fed corn starch‐based diets when compared to the normal control rats. However, white and yellow Discorea domentorum yam flour‐based diets significantly (P < 0.05) increased SOD and CAT activities, GSH and MDA levels while reduced urea and creatinine levels in treated diabetic rats but not up to control levels. The results suggest that the Discorea domentorum yam flour based‐diet may attenuate hyperglycemia, nephrotoxicity and oxidative stress in diabetic condition.

Support or Funding Information

NIL

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anti‐adipogenic and Anti‐diabetic Activities of Patchouli Alcohol
Authors: Lee Jihye , Lee Seong‐Ho
Published: June 2021

Patchouli alcohol is a sesquiterpene alcohol found in Pogostemon cablin. We reported that patchouli alcohol showed anti‐inflammatory and anti‐cancer activities in murine macrophages and human colon cancer cell lines. However, its effect on metabolism remains questionable. The current study was designed to investigate if patchouli alcohol affects adipogenesis and insulin sensitivity using differentiated adipocytes and myocytes, respectively. Treatment of patchouli alcohol (50, 75, and 100 μM) reduced lipid accumulation in 3T3‐L1 adipocytes in a dose‐dependent manner without toxicity. Treatment of patchouli alcohol increased expression of non‐phosphor and total β‐catenin in 3T3‐L1 adipocytes. On the other hand, treatment of patchouli alcohol (25 and 50 μM) significantly increased glucose uptake in differentiated C2C12 cells, which is associated with increased phosphorylation of Akt, AMP‐activated protein kinase α (AMPKα), insulin receptor substrate‐1 (IRS‐1), and β‐catenin. We demonstrated that β‐catenin might mediate beneficial effect of patchouli alcohol on prevention of adipogenesis and enhanced glucose uptake.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Obesity‐Related Stressors Repress Gonadotropin‐releasing Hormone Gene Expression via the Transcription Factor c‐Fos
Authors: Moseman Warren , Bertsch Alec , Walsh Heidi E.
Published: June 2021

In obesity, endoplasmic reticulum (ER) stress is a well‐characterized mediator of hormonal dysfunction in multiple tissues, including liver, pancreas, and adipose tissue. In the brain, ER stress induced by excess nutrients and inflammatory cytokines disrupts insulin and leptin sensitivity in hypothalamic neurons controlling food intake. Obesity‐related cellular stressors may also disrupt hypothalamic control of the reproductive system, as obesity is a risk factor for infertility in both men and women. Thus, we sought to determine how ER stress and nutrient excess directly affect production of gonadotropin‐releasing hormone (GnRH) by hypothalamic neurons. GnRH controls pituitary gonadotropin release and subsequent gonadal function, and expression of the Gnrh1 gene is regulated by multiple transcription factors and signaling pathways. Using an immortalized mouse hypothalamic cell line (GT1‐7), we determined that ER stress induced by tunicamycin or thapsigargin repressed expression of the Gnrh1 gene as measured by qRT‐PCR. We also exposed cells to a saturated fatty acid (palmitic acid) to mimic excess nutrients in obesity and observed similar Gnrh1 repression. Both ER stress and palmitic acid promoted inflammation in GT1‐7 cells, as measured by NFκB and JNK activation and IL‐6 production. Repression of Gnrh1 by ER stress was partially rescued by PKC and JNK inhibitors. Because ER stress and palmitic acid upregulated expression of Fos, and because c‐Fos is a known repressor of Gnrh1, we asked whether inhibition of c‐Fos transcriptional activity could rescue Gnrh1 repression. The c‐Fos/AP‐1 inhibitor T5224 was able to completely reverse repression of Gnrh1 by ER stress and palmitic acid, indicating a pivotal role for c‐Fos induction in Gnrh1 repression by obesity‐related stressors. Thus, our work suggests that obesity‐related stressors can directly impact GnRH neurons, and that dysregulation of Gnrh1 expression may contribute to obesity‐related infertility.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Changes in Spexin Levels are influenced by the Presence or Absence of Gestational Diabetes in Pregnant Subjects
Authors: Aldaghri Nasser
Published: June 2021

Objective

The study aimed to determine whether circulating spexin (SPX) is modified during the course of pregnancy and whether it is affected by the presence of glucose intolerance.

Methods

This prospective follow up study involved 47 pregnant women (26 non‐GDM and 21 GDM; mean age 31.0 ± 5.4 years; mean BMI 28.2 ± 5.8 kg/m2). Anthropometrics, glycemic and lipid profile as well adipocytokines and spexin were measured at baseline and after 3 and 6 months.

Results

In GDM patients, SPX levels increased significantly after 6‐months, along with the significant increase in glucose during the same time (p=0.04). In non‐GDM patients, median SPX level decreased significantly from baseline to 6‐months (p<0.01) but no such significant change in glucose was observed. Change in glucose from baseline to 6‐months was significantly and positively associated with change in SPX in GDM patients only (R= 0.45; p<0.05).

Conclusions

Changes in SPX levels seem to be influenced by the presence of glucose intolerance (GDM) among pregnant women, with decreasing levels among those without GDM, and increasing among those with GDM.

Support or Funding Information

The study was funded by Prince Mutaib Chair for Biomarkers of Osteoporosis (PMCO), Deanship of Research Chairs in King Saud University, Riyadh, Saudi Arabia

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