bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell Growth and Transglutaminase 2 Expression within the Human Erythroleukemia Cell Line (HEL)
Authors: Schultz Shannon L , Birckbichler Paul J
Published: June 2021

The objective of the study was to explore the biological responses of retinoic acid and sodium butyrate on human erythroleukemia cells (HEL). The process of apoptosis is exemplary in that the cytoskeleton collapses and the DNA is broken into fragments. The cellular components of the dead cell after undergoing apoptosis are able to be recycled. Furthermore, a cell that has experienced apoptosis does not disturb its neighbors. By learning the basics of human cell experimentation, further analysis was conducted of the HEL cell lines, such as an apoptotic assay and indirect immunofluorescence. Cells were treated with either buffer (control), retinoic acid (20 uM), sodium butyrate (100 uM) or a combination of retinoic acid (20 uM) and sodium butyrate (100 uM). Cell growth rates declined when the cells were treated with the chemicals; the greatest difference being in the combination culture of retinoic acid and sodium butyrate. The enzyme transglutaminase (TG2) is an enzyme found in large amounts within some cancer cells, and is known to have multiple functions. When the cells were treated with a monoclonal antibody to TG2, transglutaminase enzyme fluorescence in the control cultures were observed to be cytoplasmic. The retinoic acid‐treated cultures appeared to have a strong presence of the enzyme in the cytoplasm. The amount of TG2 appeared to increase in all the cultures with time with the greatest increase occurring in the combination cultures. After 144 hours a portion of the combination cultures had been converted to apoptotic bodies. Future research will attempt to localize the actual location of the transglutaminase when treated with both chemicals, and if it promotes or suppresses the viability of the cancer cells. Studies are underway to determine the amount of transglutaminase that is present in each of the samples.

Support or Funding Information

Funding provided by Department of Chemistry, Slippery Rock University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Embryonic Stem Cells Derived Exosomes Enhances Chemosensitivity of Doxorubicin in Breast Cancer Cells
Authors: Das Anindita , Hirsch Alexander , Samidurai Arun , Singla Ritu , Singla Dinender K , Kukreja Rakesh C
Published: June 2021

Doxorubicin (DOX) is the most effective anti‐tumor agent to treat wide variety of solid tumors, including the breast cancer. However, it is not known whether combination treatment of embryonic stem cells derived exosomes (ES‐exos) with DOX influences the killing of breast cancer cells. To address this question, breast cancer cell lines, MDA‐MB231 and MCF7 were treated with DOX (1mM) ± ES‐exos (5 μg/ml, 10 μg/ml or 20 μg/ml) for 24 and 48 hrs. Cell death was assessed by trypan blue exclusion assay and cell proliferation was measured by MTS assay. DOX treatment of MDA‐MB‐231 cells caused significant increase in cell death at 48 hrs post treatment, from 11.4 ± 0.01 in non‐treated controls to 38.2 ±1.2 % (p<0.05, n=4; Figure 1A). However, combinatorial treatment with DOX and ES‐exos increased cell death to 49.2± 2.1% (p<0.05 vs DOX, n=4; Figure 1A). Identical results were obtained when MCF‐7 cells were treated with DOX ± ES‐exos (Figure 1B), although these cells were relatively less sensitive to cell death compared to the MDA‐MB231 cells. Similarly, co‐treatment with ES‐exos resulted in an additive effect on DOX‐induced reduction of proliferation in both cell lines. Treatment of cancer cell lines with ES‐exos alone (concentrations ranging from 5 μg/ml to 20 μg/ml) had no significant effect on cell death or proliferation. These results suggest that ES‐exos dramatically sensitize the cell killing effect of DOX in breast cancer cells which may translate to enhancing the anti‐tumor properties of DOX. We also identified 30 to 400 fold increase in miR‐96, miR‐124, miR‐200a, b, c, miR‐205, MiR‐211 in ES‐exos compared with MEF‐exos (exosome isolated from non‐ES cells) (Figure 1C). Further investigations are in progress to demonstrate the functional role of the miRs or their protein targets in enhancing the chemosensitivity of DOX

Support or Funding Information

RO1CA221813 Figure 1

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of Ras G12V/D Mutation on Downstream Pathways Using Multiplex Immunoassays
Authors: Hwang Joseph , Saporita Anthony , Xiao Qiang
Published: June 2021

Ras proteins are members of small GTPases that function as molecular switches by alternating between inactive GDP‐bound and active GTP‐bound states. Active Ras‐GTP activate downstream pathways, including MAPK pathway by binding to Raf kinase and phosphatidylinositol 3‐kinase (PI3K) to promote cellular proliferation, survival, growth and differentiation. In the 3 decades since the discovery of RAS mutations in cancer, numerous studies have established mutant RAS as a driver of tumor initiation and progression. Oncogenic mutations in Ras are found in approximately 20% of human cancers. Furthermore, 90% of pancreatic cancers harbor K‐Ras mutations with 80% of K‐Ras mutations occurring at codon 12. RAS mutation is associated with poor prognosis due to very limited therapeutic options. To address the need for better treatment options for Ras driven cancers, the RAS Initiative was established by the National Cancer Institution. Renewed efforts in understanding Ras effectors and regulatory partners, as well as advancement in new technologies for Ras assays, are currently underway. To this end, we have developed a novel multiplex immunoassay capable of simultaneously detecting total Ras, Ras G12V, Ras G12D, phospho‐MEK1, phospho‐MEK2, B‐Raf and C‐Raf in a single well. We have tested the feasibility of using this kit on cancer derived cell lines, mouse embryonic fibroblasts, and tumor tissues. This kit was able to confirm K‐Ras G12V mutation found in the COR‐L23 cell line and N‐Ras G12D mutation found in THP‐1 cell line. Furthermore, commercially purchased colon tumor tissues characterized as Ras G12V and Ras G12D positive were confirmed using our kit. Next, we analyzed downstream pathways using commercially available immunoassay kits, such as the Multi‐pathway panel and Apoptosis panel with respect to understanding differential effect of Ras G12D vs G12V mutation. In conclusion, we have developed a novel multiplex immunoassay tool that can further our understanding of Ras mutations and its effect on downstream targets.

Support or Funding Information

MilliporeSigma Corp.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Programmed Death‐Ligand 1 in Cancer
Authors: Link Tina , Dilip Megha , Caruso Olivia , Chang Margaret , Kona Abhinav , Liu Jesse , Nathan Ajay , Singh Anita , Thomas‐Wilkinson Shiloh
Published: June 2021

Programmed death‐1 (PD‐1) is an immunoregulatory receptor on cytotoxic T cells that suppresses the immune system upon interaction with its two ligands, programmed death ligand‐1 (PD‐L1) and programmed death ligand‐2 (PD‐L2). Programmed death‐ligand 1 (PD‐L1), previously known as “CD279”, is made up of 268 amino acids with a crystal structure. It is part of the immunoglobulin superfamily with a transmembrane domain and a tail which contains two phosphorylation sites. PD‐L1 is located on the cell surface of monocytes, B cells, T cells, and dendritic cells, and works in conjunction with PD‐1 to modulate an immune response. In binding with its ligand PD‐1 on an activated T cell, PD‐L1 suppresses the immune response of the T cell, rendering it inactive. PD‐L1's other ligand, the PD‐L2, also plays a role in deregulating the T cell immune response. Many cancer cells express PD‐L1, attenuating immunity and allowing cancer cell proliferation. This distorted relationship provides a focal point for cancer research. Attempts to inhibit the effects of PD‐L1 are concentrated on blocking its interaction to PD‐1, resurrecting the immune response, and thereby allowing CD8+ cytotoxic cells to attack tumor cells. Potential treatments include the development of an anti‐PD‐L1 antibody therapy to induce tumor remissions for patients with different types and stages of cancer. Supporting promising PD‐L1 research, the FDA has approved the PD‐L1 biomarker to accelerate investigation into a potential cure for cancer. In 2018, James P. Allison and Tasuku Hojo received the Nobel Prize in Medicine for research on PD‐L1 that lead to the discovery of checkpoint inhibitors that release immune responses, thereby reactivating T‐cells. The Walton High School MSOE Center for Biomolecular Modeling SMART Team has designed a 3‐D model of PD‐1 in interaction with its ligand PDL‐1 using JMOL to investigate the relationship between the structure and function of the proteins in relation to its current role in cancer development and its future in potential treatment.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Experimental Modal Analysis of Tumorigenesis and Cancer Metastasis
Authors: Martinez Bridget , Yang Yongchao , Farrar Charles , Mukundan Harshini , Nath Pulak , Mascareñas David
Published: June 2021

Traditionally, performing an experimental modal analysis of a building/structure required instrumenting the structure with a spatially distributed array of accelerometers or strain gages. Alternatively, a laser doppler vibrometer would have to be scanned across the structure of interest in a sequential manner to measure structural response. Recently, researchers at LANL developed a technology that combines the theory of structural dynamics with computer vision that provides the capability to characterize structural dynamics at very high spatial density using only an imager. With this newfound success at the macro‐scale, we have exploited this novel technology to a whole new scale‐to studying the basic structure of life itself, the human cell. We hypothesize that this new technology and novel application will provide a significantly better understanding of how stiffness and mass distribution changes in a cell as it undergoes epithelial‐mesenchymal transition, and in identifying its associated EMC biochemical cues, highlight potential therapeutic targets. For the first time it should be possible to measure the high‐resolution mode shapes of cells; given that all cells undergoing cancer metastasis experience a breakdown in the cytoskeleton, this work will enable groundbreaking advances. It is imperative to highlight, that we are only beginning to understand the relationship between biophysical properties of cells and their potential to regulate tumorigenesis and motility. This knowledge could be used to provide verification and validation of finite element models of cellular structure. This work will represent the first time that expertise in experimental structural dynamics will be brought to bear on the problem of characterizing the structural dynamics of cells at high spatial resolution, which is novel and unique on its own. When successful, this new technology could be used to couple the biophysical cues associated with other detrimental human pathologies.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protease‐activated receptor 2 (PAR‐2) biased agonism in ovarian cancer progression
Authors: Pawar Nisha R. , Duru Nadire , Conway Gregory D. , Antalis Toni M.
Published: June 2021

Ovarian cancer is one of the most fatal gynecological malignancies due to its delayed clinical presentation, indicating a need to investigate molecular mechanisms of advanced disease. Dysregulated proteolysis contributes to crucial steps of ovarian cancer metastasis, including shedding and dissemination of cells to the peritoneal cavity. Several studies have identified membrane‐anchored serine proteases, namely testisin and matriptase, that are aberrantly overexpressed in ovarian cancer. However, detailed mechanisms of activation, regulation, and downstream signaling of these proteases in this context are unknown. They have been shown to cleave and activate protease‐activated receptor‐2 (PAR‐2), a seven‐transmembrane G‐protein‐coupled receptor (GPCR). PAR‐2 has roles in hemostasis, inflammatory responses, and angiogenesis, and its overexpression has been correlated with advanced‐stage ovarian cancer. PAR‐2 has been shown to preferentially activate signaling pathways with different protease agonists, known as “biased agonism”, which can trigger different biological processes. Our overall goal is to understand and compare necessary components of testisin and matriptase activation of PAR‐2 and how resulting signaling regulates ovarian cancer progression. We hypothesize that testisin and matriptase differentially modulate PAR‐2 on the cell surface to promote cell signaling bias leading to alternative regulation of ovarian tumor progression. We are currently characterizing human ovarian cancer cell lines with stable overexpression of testisin or matriptase to compare the effects on migration, invasion, and other processes of tumor progression including metastasis using a mouse model of ovarian cancer. Interfering with these pathways might provide unique opportunities for the development of new mechanism‐based strategies for the treatment of advanced ovarian cancers.

Support or Funding Information

NIH R01 CA196988, Cancer Biology T32 Training Grant (University of Maryland Baltimore School of Medicine)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biochemical Basis of Steroid Hormones Inhibition of Human Melanoma Cell Growth In‐Vitro & Its Implication on Melanoma Treatment
Authors: Ramaraj Pandurangan
Published: June 2021

Skin, as an endocrine organ produced various hypothalamic, pituitary, adrenal and sex steroids. Steroids were not only important for healthy skin, but were also shown to influence locally and systemically. This raised the question whether melanoma was a hormone‐dependent cancer. But to the contrary, clinical study showed that menstruating females were better protected in melanoma than post‐menopausal women and men of any age. Literature showed that progesterone (P) level varied in menstruating females between 1000 to 1500 ng/dL. Whereas in post‐menopausal women, progesterone ranged between 20 to 100 ng/dL and in males between 27 – 90 ng/dL. In addition, our in‐vitro research showed that progesterone significantly inhibited human melanoma (BLM) cell growth. So, it was hypothesized that progesterone could be protecting menstruating females in melanoma. Further research showed that androgens (androstenedione, AD and testosterone, T) also inhibited human melanoma cell growth in‐vitro, but only at high concentrations (100 μM and 200 μM). Co‐incubation of AD and T separately with P showed an additive effect on BLM cell growth inhibition, suggesting the protective function of P. In order to find out the biochemical basis of steroid hormones action, Elisarray was carried out on the supernatants. There was a specific suppression of IL‐8 cytokine in the supernatants of AD, P and AD+P co‐incubated cells compared to untreated control BLM cells. Same experiments were repeated on another human melanoma (1205Lu) cell line, which also showed a specific suppression of IL‐8 cytokine. The decrease in IL‐8 was quantitated by Elisa, which correlated with the decrease in melanoma cell growth. To further confirm, experiments with T, P and T+P were carried out on BLM and 1205Lu cell lines, which also showed a specific suppression of IL‐8 by Elisarray. In addition, Kanda and Watanabe already showed that dihydrotestosterone and estrogen also suppressed IL‐8 in melanoma cells.

Conclusion

Suppression of IL‐8 resulting in the suppression of melanoma cell growth, implied the important role of IL‐8 in regulating melanoma growth. But suppression of IL‐8 in turn by various steroids, indicated the local regulation by the steroids. Local regulation in turn indicated the role of steroids in regulating melanoma growth. Hence, balancing or restoring local steroid hormones along with targeted or immuno therapy could be an ideal treatment for melanoma.

Support or Funding Information

1) Start‐up grant to PR2) Biomedical Sciences research grant3) Warner/Fermaturo research grant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Potential Block of Direct and Secondhand Smoke‐Induced Increase in Endothelin‐1 Signaling of Breast Cancer Cell Lines by Hinute‐AM
Authors: Finch Jordon , Mukherjee Shyamali , Hussein Nimo , Maebuchi Motohiro , Das Salil K
Published: June 2021

Several epidemiological studies suggest that cigarette smoking as well as exposure to second‐hand smoke increase the incidence of breast cancer. Diet also plays an important role in breast cancer as well as other diseases. However, few studies have examined the molecular mechanisms by which cigarette smoke and diet alter breast cancer incidence. The neuropeptide hormone endothelin‐1 (ET‐1) has been identified as a multifunctional molecule that promotes cancer growth. High levels of ET‐1 expression are also associated with reduced survival of breast cancer patients. The goal of the present study was to assess whether direct or passive exposure to cigarette smoke alters the expression of ET‐1 and one of its receptors, ETAR, in non‐tumorigenic breast cells and breast cancer cell lines. To determine whether dietary factors alter this increase in ET‐1 and ETAR, cells were also exposed to Hinute‐AM, a commercially marketed soy protein hydrolysate. Proliferation assays were used to measure the effect of cigarette smoke on non‐tumorigenic breast cells. Both direct and second hand smoke exposure increased proliferation of MCF‐10, MCF‐12A and MCF‐12F non‐tumorigenic breast cell lines. We next performed Western blot analysis to test the effect of cigarette smoke of ET‐1 and ETAR expression. Exposure to direct and second‐hand cigarette smoke increased ET‐1 and ETAR protein within non‐tumorigenic MCF‐10A cells as well as BT549 and MCF‐7 breast cancer cells. Low concentrations (0.02–0.20 mg/mL) of Hinute – AM reduced proliferation of breast cancer cells and protein expression of ET‐1. In conclusion, our data suggest that cigarette smoke may increase the proliferation and aggressiveness of breast cancer by increasing expression of ET‐1 and its receptors. Furthermore, it may be possible to counteract the effects of cigarette smoke with dietary supplements such as Hinute‐AM.

Support or Funding Information

[Supported by Grants from Army (DAMD 17‐03‐1‐0352), FAMRI (Clinical Innovator Award 062415), Fuji Oil Company, Osaka, Japan, NCI (R25CA214220) and NIH (MeTRC5U5AMD007593).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluating the impact of bead media diameter and material composition on bacterial cell lysis and genomic DNA extraction
Authors: Proctor Caleb , Soldat Shelby M , Easparro Brandon , Nash Rodney , Atwood James
Published: June 2021

Mechanical lysis of bacteria is a common alternative to enzymatic, detergent, or tonicity‐based methods. Mechanical lysis is needed in areas of research where introducing chemicals can compromise either the assay or the analyte and is achieved in a variety of ways, one of which is bead beating. Bead beating utilizes small beads of various sizes and composition to contact the sample with high energy to facilitate lysis. This type of lysis can be achieved using many different types of equipment from laboratory vortexers to specialized bead mills. Maximizing the amount of lysis is important for both consistency of analyte extractions and increasing yield when sample size is limited.

Herein, we evaluate two bead media compositions (borosilicate glass and yttria‐stabilized zirconium oxide) and two sizes of each of those compositions (0.5 mm and 0.1 mm) to analyze what factors are most important to achieve the maximum amount of lysis using cultures of Escherichia coli, Staphylococcus epidermidis, and Nocardia brasiliensis. Speed, time, number of beads, and mass were all controlled to identify the ideal size, composition, force and duration to maximize cell lysis while maintaining nucleic acid integrity. In order to evaluate lysis efficiency cells were disrupted then plated to enable colony counting. After each processing step, genomic DNA was purified, quantified, and analyzed by gel electrophoresis to evaluate genomic DNA integrity.

In all cases, it was determined that bead diameter played a greater role in lysis efficiency when compared to bead material even though kinetic energy of the zirconium oxide beads is 2X greater than the glass at a given tube velocity. E. coli was lysed at greater than 90% total cell lysis in three minutes at a tube velocity of 4.2 m/s (Figure 1). No DNA degradation was observed after homogenization for 1 minute. Increasing amounts of DNA shearing was observed as processing was increased (Figure 2).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Symbiosis and Host Immunity in Reef Ecosystems
Authors: Lazar Stephen , Palmer Andrew G
Published: June 2021

Many marine reef‐building corals live in nutrient poor conditions, forming symbiotic relationships with dinoflagellates of the genus Symbiodinium to supplement or replace active feeding. In order to maintain this symbiotic state, and avoid being consumed or expelled, these symbionts suppress the immune system of the host. Yet these corals do not show high levels of disease under standard conditions. The simplest hypothesis is that Symbiodinium are able to compensate for this reduction in immunity. Many common reef diseases are associated with bacteria which regulate the transition from being benign to virulent through the population detecting process of quorum sensing (QS). Other algae have proven capable of metabolizing the N‐acyl‐L‐homoserine lactone (AHL) class of QS signals, potentially disrupting these events. In this study we have investigated the ability of members of the genus Symbiodinium to regulate bacterial quorum sensing as a strategy for supplementing the host immune system.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis and Biological Evaluation of a Photoswitchable Quorum Sensing Molecule
Authors: Karns Taylor , Stajdl Shelby , Zimmer Kassidy , Rohlman Christopher , Streu Craig
Published: June 2021

Quorum sensing is the ability of bacteria to communicate with one another via chemical signals. This ignites a response within the collective group of bacteria to produce a single response that ultimately benefits the bacteria, an example being biofilm formation. If this natural process could be controlled or coopted, the biotecnical applications arising from this natural ability of bacteria could be limitless. Photoswitching molecules are able to respond to a light stimiulus. Azostilbenes in particular isomerize from trans to the cis conformation in response to the appropriate wavelengths of light. If one of the available conformations is bioactive, while the other is not, this photoisomerization has the effect of activating or inactivating the molecule in response to light. The power of light responsive chemical signaling systems comes from their ability to achieve exquisite spatial and temporal control, which is of tremendous utility for numerous bioindustrial applications. To this end, we have designed a series of photoswitching molecules based upon N‐acyl homoserine lactones (AHLs), which are known to be prominent chemical messengers in numerous bacterial species, by integrating the azo‐stilbenoid motif into the parent molecule scaffold. These molecules are made by Mills coupling between the appropriate aniline and aryl‐nitroso species. Their photokinetic properties are measured by UV/Vis spectroscopy and their bioactivity measured in the presence and absence of isomerizing light. We herein describe the synthesis, photokinetic studies, and bioactivity of this class of photoswitchable molecule. We also describe potential industrial applications of this technology.

Support or Funding Information

Albion College Foundation for Undergraduate Research, Scholarship, and Creative Activity

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of Prostate Cancer Cell Proliferation and Survival by Rosemary Extract
Authors: Jaglanian Alina , Tsiani Evangelia
Published: June 2021

Prostate cancer is the most commonly diagnosed cancer in men. Despite many advances in diagnosis and treatment, prostate cancer is the second‐leading cause of cancer related death amongst men in North America and therefore there is an urgent need to find novel chemicals that can be used effectively towards the treatment of prostate cancer patients. Prostate cancers are typically characterized by the expression of androgen receptor (AR) and prostate‐specific antigen (PSA). Prostate cancers that are AR positive can be treated with hormonal therapy, however those which are AR negative are more aggressive and do not respond to hormone therapy, thus this subtype of prostate cancer requires targeted treatments. Many chemotherapeutic drugs have been discovered by screening natural products from plants. Polyphenols derived from the plant rosemary, have been found to have antioxidant and anti‐inflammatory effects and are proposed to have anticancer activity. In previous studies, rosemary extract (RE) has been found to reduce cell proliferation, viability, survival, and increase apoptosis in various cancer cell lines. However, there is very limited evidence examining RE's effects in androgen receptor negative prostate cancer cells. In the present study, we examine the effects of rosemary extract on PC‐3 prostate cancer cell proliferation, survival, migration and apoptosis. RE significantly inhibited PC‐3 cell proliferation and survival in a dose dependent manner. Furthermore, RE reduced cell migration and enhanced the cleavage of PARP, a marker of apoptosis. Our findings indicate that RE may have potent anti‐cancer properties against AR negative prostate cancer and supports the need for further investigation into its role in modulating key signaling pathways involved in cell proliferation, survival, and migration.

Support or Funding Information

Supported by the Prostate Cancer Fight Foundation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of Polyamine Transport Inhibitors in a Drosophila Epithelial Model
Authors: von Kalm Laurence , Wang Minpei , Phanstiel Otto
Published: June 2021

Increased polyamine biosynthesis activity and an active polyamine transport system are characteristics of many cancer cell lines and polyamine depletion has been shown to be a viable anticancer strategy. Polyamine levels can be depleted by difluoromethylornithine (DFMO), however, malignant cells frequently circumvent DFMO therapy by up‐regulating polyamine import. Therefore, there is a need to develop compounds that inhibit polyamine transport. We have previously shown that the pattern of uptake of a series of polyamine analogues in a Drosophila model epithelium shares many characteristics with mammalian cells, indicating a high degree of similarity between the mammalian and Drosophila polyamine transport systems. Here, we focused on the utility of the Drosophila epithelial model to identify and characterize polyamine transport inhibitors. We show that a previously identified inhibitor of transport in mammalian cells has a similar activity profile in Drosophila. The Drosophila model was also used to evaluate two additional transport inhibitors. We further demonstrate that a cocktail of polyamine transport inhibitors is more effective than individual inhibitors, suggesting the existence of multiple transport systems in Drosophila. Our findings reinforce the similarity between the Drosophila and mammalian transport systems and the value of the Drosophila model to provide inexpensive early screening of molecules targeting the polyamine transport system.

Support or Funding Information

University of Central Florida, College of Sciences Seed Award

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Knockout of Fatty Acyl‐CoA Synthetase ACSVL3 in Glioma Cells Produces Diverse Metabolic Alterations in Non‐Lipid Pathways
Authors: Watkins Paul , Shi Xiaohai , Kolar Elizabeth , Clay Emily , Liu Yanqiu
Published: June 2021

Knockout (KO) of the fatty acid‐activation enzyme very long chain acyl‐CoA synthetase 3 (ACSVL3; SLC27A3) in glioblastoma cells reduced their malignant growth properties both in vitro and in xenografts. KO U87 glioma cells grew at a slower rate, became adherence‐dependent, and were less invasive than parental U87 cells. KO cells produced fewer, slower‐growing subcutaneous and intracranial tumors when implanted in NOD‐SCID mice. To understand the mechanisms underlying these changes, we investigated several possibilities. We first hypothesized that lack of ACSVL3 might reduce the rate of incorporation of labeled fatty acids into membrane phospholipids; however, no differences were observed. We then investigated the possibility that lack of ACSVL3 affected either apoptosis or autophagy, but detected no differences in these processes between control and KO U87 cells. We next examined cell cycle, and found that KO produced significant changes. In non‐synchronized cells, parental U87 cells were mainly in G0/G1, while ACSVL3 KO cells were mainly in S‐phase. After synchronization by serum starvation, both U87 and KO cells started out mainly in G0/G1, but by 12 hours ACSVL3 KO cells shifted significantly toward S‐phase. Consistent with this finding, the levels of several cyclins and cyclin‐dependent kinases were found to be altered in KO cells. In addition to effects on lipid metabolism, proteomic and metabolomics profiling indicated that ACSVL3 KO produced changes in glucose and energy metabolism. Even though protein levels of glucose transporters GLUT1 and GLUT3 were reduced by KO, cellular uptake of labeled 2‐deoxyglucose was unaffected. Glucose oxidation to CO2 was reduced 40% by ACSVL3 depletion, and the cellular glucose level was 25% higher in KO cells. Glycolytic enzymes were upregulated by KO, but metabolic intermediates were essentially unchanged. Surprisingly, lactate production and the levels of both LDHA and LDHB were elevated by ACSVL3 KO. The activity of the pentose phosphate pathway was found to be lower in KO cells. Citric acid cycle enzymes, electron transport chain complexes, and ATP synthase protein levels were all reduced by ACSVL3 depletion. Mitochondria were elongated in KO cells, but had a more punctate morphology in U87 cells. We conclude that beneficial effects of ACSVL3 depletion in human glioblastoma cells are the result of alterations in diverse metabolic processes that are not directly related to fatty acid and/or lipid metabolism.

Support or Funding Information

Supported by Institutional Funds.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis and characterization of BODIPY‐FL‐cyclosporine A as a substrate for both human and mouse multidrug resistance‐linked P‐glycoprotein
Authors: Sajid Andaleeb , Natarajan Raju , Lusvarghi Sabrina , Vahedi Shahrooz , Swenson Rolf , Ambudkar Suresh V
Published: June 2021

Generation of fluorescent conjugates of small molecules and existing drugs has facilitated their use in addressing cellular functions and pharmacological analysis. These compounds interact with several proteins as substrates or inhibitors, thus allowing the development of unique fluorescence‐based methods to study in vivo localization and the molecular mechanisms. Pglycoprotein (P‐gp) is an ATP‐binding cassette transporter that effluxes most anti‐cancer drugs from cells, contributing to the development of drug resistance in cancer cells. P‐gp, which exports toxic metabolites and xenobiotics, is expressed on the surface of epithelial cells of the intestine, kidney, liver, placenta, adrenal gland and endothelial cells at blood‐brain barrier. With the aim of developing an effective probe to study drug‐transport by P‐gp in various model systems including cultured cells, mice and zebra fish, we synthesized a BODIPY‐FL conjugate of cyclosporine A (BD‐CsA). After synthesis and characterization of its chemical purity, BD‐CsA was compared with the currently available 7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl (NBD)‐CsA probe in terms of its usage as a substrate of human and mouse P‐gp. Using a BacMam baculovirus HeLa cell expression system and a flow cytometry‐based transport assay, we found that BD‐CsA is recognized as a substrate by both human and mouse P‐gp. The rate of efflux of BD‐CsA by human P‐gp is similar to that of NBD‐CsA (T1/2 of 5.0 min and 3.0 min for BD‐CsA and NBD‐CsA, respectively). In flow cytometry assays, the fluorescence intensity of BD‐CsA was almost 10‐times higher than that of NBD‐CsA due to the high fluorescence quantum yield of the BODIPY fluorophore, allowing us to use significantly lower concentrations of BD‐CsA to achieve the same fluorescence levels. The transport of BD‐CsA was inhibited by the P‐gp inhibitor tariquidar, with similar IC50 values as for NBD‐CsA transport (21.6 nM for BD‐CsA and 25.8 nM for NBD‐CsA). We also observed that both BD‐CsA and NBD‐CsA partially inhibited the ATPase activity of P‐gp. In silico docking of BD‐CsA and NBD‐CsA to the homology model of human P‐gp indicates that both probes bind in the drug‐binding pocket of Pgp with similar docking scores. Thus, we demonstrate that BD‐CsA is a sensitive and efficient fluorescent substrate of P‐gp that can be used to study the transporter's function in both in vitro and in vivo settings.

Support or Funding Information

This research was funded by the Intramural Research Program of the National Institutes of Health, the National Cancer Institute, Center for Cancer Research

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Function of the KCNQ1 Potassium Ion Channel in the Human Heart
Authors: Link Tina , Jacobson Reagan , Lee Chase , Maraghi Sophie , Mohan Siddharth , Shabazaz Tara , Vemana Riya , Wright Aryn
Published: June 2021

KCNQ1 is a homotetrameric voltage‐gated potassium ion channel subunit protein. KCNQ1 is the alpha‐subunit of the ion channel and forms heteromultimers with protein members of the KCNE family (“beta subunits”) to create a pore in the membrane of a cell. Two of these beta subunits are KCNE1 and KCNE3. Under physiological conditions, four KCNQ1 alpha subunits combine with two or four beta subunits to form the potassium ion channel. The alpha subunits form the structure of the channel whereas the beta subunit binds onto the channel and regulates its activity. KCNQ1 is embedded in the membranes of numerous types of cells, including cardiac myocytes, epithelial cells, and cells in the human inner ear. In human cardiac myocytes, KCNQ1 and KCNE1 work to regulate the rhythm of the heart by controlling the repolarization phase of the cardiac action potential. Almost three hundred mutations have been identified in the KCNQ1 gene, which is located on chromosome 11. The majority of these are missense mutations and alter the protein's structure to the extent that the flow of potassium ions out of the cardiac cells is disrupted. The most common condition resulting from a mutation in KCNQ1 in hereditary Long QT Syndrome (LQTS), which is characterized by a delay in the ventricular repolarization component of a heart beat. Symptoms associated with LQTS include dizziness, fainting, and potentially seizures and sudden death. In rare cases, a mutation in KCNQ1 can also cause further complications, which include atrial fibrillation, Short QT syndrome, and Jervell and Romano‐Ward syndrome. Treatments are available for LQTS, such as making lifestyle changes, taking beta blockers, and implanting a defibrillator. Current research in medication for LQTS is centered around compounds such as hexachlorophene that can target the individual subunits in the ion channel as opposed to the entire channel. Pairing compounds with this ability may provide enough correction to the subunits that LQTS can be effectively “cured.” The Walton High School SMART Team has designed a 3D model of the KCNQ1 ion channel using JMOL to investigate the relationship between structure and function.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Podocyte‐Specific Rescuing and Deletion of Acid Ceramidase Gene in Mouse Glomeruli
Authors: Zhang Qinghua , Li Guangbi , Yuan Xinxu , Bhat Owais M , Lohner Hannah , Li Pin‐Lan
Published: June 2021

We have recently developed a podocyte‐restricted acid ceramidase (AC) gene knockout mouse model (Asah1fl/fl/Podocre, Asah1 is a code of mouse AC gene), which was shown to produce podocyte dysfunction and consequent minimal change disease (MCD). The present study attempted to develop somatic gene intervention methods to perform podocyte‐specific gene deletion, rescuing and silencing of this AC gene in Asah1fl/fl/Podocre mice and their control littermates including Asah1fl/fl/WT mice, which could exclude the potential development or compensatory influence given that Asah1 gene was embryonically deleted in podocytes in those animal strains. We first constructed 2 different vectors which can be transfected to change AC gene expression including a podocyte‐specific AC gene (podo‐Asah1) expression vector and a podocyte‐specific Cre gene (podo‐Cre) expression vector. The pEZX vector containing podocin promoter was used to generate Asah1 and Cre full length gene expression plasmids, which were transfected into Asah1fl/fl/Podocre mice to rescue Asah1 gene and into Asah1fl/fl/WT mice to delete floxed Asah1 gene, respectively. Using isolated podocytes from 2 different mouse strains, we confirmed by immunofluorescence staining, real‐time PCR and Western blot analysis that these vectors could be successfully transfected into podocytes and produced corresponding effects to alter Asah1 gene expression, for example, rescuing of this gene in Asah1fl/fl/Podocre podocytes and deletion of this gene in Asah1fl/fl/WT podocytes. In animal experiments, we used microbubble ultrasound transfection techniques to introduce different plasmids into the kidneys. It was found that Asah1 gene was rescued in podocytes of Asah1fl/fl/Podocre mice by podo‐Asah1 plasmids and deleted in Asah1fl/fl/WT mice by podo‐Cre plasmids. Functionally, we demonstrated that increased Asah1 expression in podocytes decreased desmin levels, but enhanced podocin expression, indicating restoration of podocyte function. It also enhanced the interaction of multivesicular bodies (MVBs) and lysosomes as shown by increase in Vps16 and Lamp‐1 colocalization, which resulted in decreased exosome release from podocytes. Taken together, our results suggest that somatic transfection of podocin promoter‐driven Asah1 and Cre gene expression vectors produced podocyte‐restricted gene changes in different gene engineered animal models for AC function studies.

Support or Funding Information

supported by NIH grant DK54927, DK120491 and HL057244

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acid Sphingomyelinase Deficiency Protects Mitochondria and Improves Function Recovery after Traumatic Brain Injury
Authors: Novgorodov Sergei A. , Voltin Joshua R. , Riley Christopher L. , Gudz Tatyana I.
Published: June 2021

Traumatic brain injury (TBI) is one of the leading causes of disability worldwide and a prominent risk factor for neurodegenerative diseases. The expansion of nervous tissue damage after the initial trauma involves a multifactorial cascade of events including excitotoxicity, oxidative stress, inflammation and deregulation of sphingolipid metabolism that further mitochondrial dysfunction and secondary brain damage. Our studies establish a fundamental role of acid sphingomyelinase (ASM) in the multi‐faceted mechanisms governing secondary brain injury after TBI. The data indicate that TBI‐triggered activation of ASM and upregulation of mitochondrial sphingosine resulted in mitochondrial respiratory chain malfunction leading to an enhanced assembly and activity of the NLRP3 inflammasome, a pivotal factor promoting the neuroinflammatory response to brain injury. The results of our studies provide further support for the emerging role of ASM in repressing the cell maintenance and quality control mechanism, autophagy, which is essential for the neural cell survival after the injury. Thus, knocking down ASM rescued mitochondrial defect, augmented the autophagic flux, downregulated the neuroinflammation and improved the brain function recovery after TBI. Importantly, ASM is involved in the initial stages of progression of astrogliosis, a brain defense mechanism wherein activated astrocytes minimize spread of the neuroinflammation and further tissue repair and regeneration in response to TBI. Our novel findings reveal that ASM, a critical player promoting secondary brain injury and neuroinflammation is uniquely positioned to be a promising target for the development of effective pharmacological treatment of patients with TBI.

Support or Funding Information

Supported by NIH grant R01NS083544 (TIG) and VA Merit Award I01BX002991 (TIG).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ULK1‐dependent Mitophagy Is Essential for Maintaining Cardiac Function during High Fat Diet‐induced Diabetic Cardiomyopathy
Authors: Tong Mingming , Saito Toshiro , Zhai Peiyong , Oka Shinichi , Sadoshima Junichi
Published: June 2021

Diabetic patients develop cardiomyopathy characterized by hypertrophy, diastolic dysfunction, lipotoxicity, and mitochondrial dysfunction. How mitochondrial function is regulated in diabetic cardiomyopathy remains poorly understood. Mice were fed either a normal diet (ND) or a high fat diet (HFD, 60 kcal % fat). Mitophagy, evaluated with Mito‐Keima, was increased after 3 weeks of HFD feeding (mitophagy area: 8.3% per cell with ND and 12.4% with HFD) and continued to increase after 20 weeks (p<0.05). Although we have shown recently that mitophagy during the early phase of HFD feeding is mediated by Atg7‐dependent mechanisms, the mechanisms mediating mitophagy in the heart during the chronic phase of HFD feeding remain poorly understood. Phosphorylation of ULK1 was activated and Rab9 protein level was increased in the mitochondrial fraction within 20 weeks of HFD consumption (p<0.05). By isolating adult cardiomyocytes from GFP‐Rab9 transgenic mice fed HFD, we discovered that mitochondria were sequestrated by Rab9‐positive ring‐like structures. Since ULK1 regulates Rab9‐dependent mitophagy, we fed ULK1 cKO mice with HFD for 20 weeks. In wild type (WT) mice, cardiac hypertrophy and diastolic dysfunction (EDPVR = 0.051±0.009 in ND and 0.115±0.006 in HFD) were induced after 20 weeks of HFD feeding (p<0.05). By crossing Tg‐Mito‐Keima mice with ULK1 cKO mice, we found that downregulation of ULK1 impaired mitophagy in response to ND or 20 weeks of HFD consumption (p<0.05). Deletion of ULK1 exacerbated diastolic dysfunction (EDPVR=0.115±0.006 in WT and 0.162±0.021 in ULK1 cKO, p<0.05) and even induced systolic dysfunction (ESPVR=22.74±2.13 in WT and 16.78±2.12 in ULK1 cKO, p<0.05) during HFD feeding. Electron microscopic analyses indicated that the mitochondrial cristae structure was disrupted more severely in ULK1 cKO mice with HFD feeding than control mice (p<0.05). In summary, genetic disruption of ULK1‐Rab9‐dependent mitophagy during the chronic phase of HFD feeding exacerbates mitochondrial dysfunction, thereby facilitating the development of diabetic cardiomyopathy. ULK1‐Rab9‐dependent mitophagy serves as an essential quality control mechanism for cardiac mitochondria during HFD feeding.

Support or Funding Information

The project was supported by AHA and NIH (5R01HL138720‐02).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mean Platelet Volume in Patients with Regulated and Unregulated Type 2 Diabetes Mellitus According to Sex
Authors: Kama Tabitha Supriya , Nayakanti Abraham Ratna J , CS Jaya Prakash , Srinivasan KR , Koteswary R
Published: June 2021

Type 2 diabetes mellitus(DM) is a metabolic disorder with a prevalence of 8.8% in adults in India and 425 million cases worldwide. The high prevalence and morbidity make it a worldwide health problem. Type 2 DM is a part of metabolic syndrome which comprises of hypertension, dyslipidemia, increased procoagulation factors and impaired fibrinolysis. HbA1c is a Glycated hemoglobin is a form of hemoglobin that is measured primarily to identify the three‐month average plasma glucose concentration in diabetics and is measured by automated ion‐exchange high performance liquid chromatography (Bio‐Rad Variant II, Hercules, CA). Hyperglycemia in diabetic patients is thought to have a harmful effect on the blood vessels. Platelet volume is a marker of platelet function and activation. It can be quantified as mean platelet volume (MPV) by clinical hematology analyzers. Platelets are involved in homeostatic process and have an important role in atherosclerosis and arterial thrombosis. When vascu‐lar injury occurs, platelets adhere to dam‐aged endothelium to form platelet plug. It has been reported that plate‐lets from diabetic patients synthesize more thromboxane than normal platelets. To compare the MPV between regulated (HbA1c<6.5%) and nonregulated (HbA1c > 6.5%) diabetics.

MATERIALS AND METHODS

Study design‐ Cross sectional study, Study duration‐ 06 months; February 2018–July 2018. Sample size‐ Based on the article the proportion used to measure the parameter MPV is 8.8% with an alpha of 5 % the corresponding z value is ‐1.95996398454005. Using the below formula with the minimum percentage difference to be deemed clinically significant as 4.7%. The study outlines the importance of MPV in regulated and non regulated type 2 DM.

Support or Funding Information

ASIP

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.