bluuebunny/crossref_metadata_2025 huggingface dataset:
Spatiotemporal Control of Glycolysis Modulates ATP Generation and Enhances Restoration of Endothelial Barrier Function Following Inflammatory Injury
Authors: Gajwani Priyanka A , Wang Li , Ray Anne Marie A , Krantz Sarah A , Karginov Andrei A , Rehman Jalees
Published: June 2021

Background

Acute inflammatory injury is characterized by dysfunction of the vascular endothelium, resulting in disruption of endothelial adherens junctions, increased vascular permeability and leukocyte infiltration. While the mechanisms of assembly and disassembly of endothelial adherens junctions have been extensively studied, little is known about the bioenergetics of the barrier repair process. The glycolysis regulatory enzyme PFKFB3 has recently been identified as a key mediator of endothelial metabolism and function and could thus serve as a potential mediator of barrier repair.

Methods and Results

To investigate how the endothelial metabolic profile changes during inflammation, we performed a Seahorse bioenergetic flux assay on Human Lung Microvascular Endothelial cells (HLMVECs). Glycolysis, as measured by the extracellular acidification rate, increased by 36% following treatment with the inflammatory mediator TNFα, while oxidative phosphorylation was not affected. Pharmacological inhibition of PFKFB3 abolished TNFα mediated upregulation of glycolysis. PFKFB3 activity was also necessary for barrier restoration of HLMVECs following injury, as measured by a transendothelial electrical resistance (TER) assay (p<0.0001). Whole cell ATP levels, measured by live cell microscopy using the fluorescent ATP biosensor Perceval HR, increased 31% following TNFα treatment (p<0.0001), largely concentrated at the cell periphery. We hypothesized that this regional ATP generation is PFKFB3 dependent. To address this question, we engineered a novel construct in which PFKFB3 is targeted to the cell membrane in an inducible manner and thus regulating metabolism in a spatiotemporal manner. Importantly, PFKFB3 membrane targeting enhanced endothelial barrier restoration by increasing the extent and rate of recovery (p<0.0001) following barrier disruption.

Conclusions

This data suggests that spatial and temporal regulation of PFKFB3 mediated glycolysis is important for restoration of the endothelial barrier after inflammatory injury. A better understanding of the endothelial barrier bioenergetics could allow for the development of novel therapeutic approaches to treat diseases in which endothelial barrier function is acutely compromised.

Support or Funding Information

AHA 18PRE34070092 National Institute of Health P01HL060678

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ganoderma lucidum extract decreases motility and lamellipodia formation of triple‐negative metastatic breast cancer
Authors: Acevedo‐Diaz Ariana C , Ortiz‐Soto Gabriela , Suarez Ivette J , Zayas Astrid , Martinez‐Montemayor Michelle M
Published: June 2021

Breast cancer (BC) is the leading cause of cancer among women in the United States and Puerto Rico. Triple‐negative breast cancer (TNBC) is characterized by the absence of estrogen and progesterone receptors, and lack of HER2 overexpression. Approximately, 15% of all women with TNBC typically display shorter overall survival and an early peak of distant recurrences at 3y after diagnosis. Treatments for TNBC are limited; currently, there are no approved targeted therapies besides non‐targeted chemotherapy. The mortality of TNBC could result from the lack of effective treatments to decrease the migration of cancer cells to other tissues. Hence, it its highly important to understand these migration processes and to find effective therapies. Therefore, we aimed to test the anti‐migration effects of the medicinal mushroom Ganoderma lucidumextract (GLE) on TNBC cell motility and on the regulation of proteins involved in this process. First, MDA‐MB‐231 cells were treated with various GLE concentrations and evaluated for cell viability and a washout assay. To study the effect of GLE on cell migration, cells were treated with vehicle, 0.25 or 0.96mg/mL (IC50) of GLE for 24h, followed by wound healing and invasion assays. The activity of Rac was tested by a pulldown activity assay and immunoblotting. Furthermore, we tested the expression of proteins important in the dynamics of cell motility Cdc42, Lamellipodin (Lpd), FAK, WAVE and Rho. The localization and expression of Lpd was visualized by immunocytochemistry. Results showed that GLE significantly decreased the cell viability on TNBC, with no cell recovery after a 72h period of treatment withdrawal. GLE significantly decreased MDA‐MB‐231 cell migration and invasion, while reducing the activity of Rac. The expression of Lpd, FAK, WAVE, c‐myc, and Cdc 42 were also modulated upon GLE treatment. GLE decreased Lpd localization in ruffled edges of cancer cells, as well as changes in nuclear and cell morphology. In conclusion, our results suggest the potential of GLE as a natural anti‐TNBC migration agent.

Support or Funding Information

This work was supported by grants from the National Institutes of Health GM111171 (MMM), MD007583 (MMM), GM103475 INBRE‐Supplies for Graduate Students (GOS), from the Puerto Rico Science, Technology and Research Trust (PRSTRT) SGRP 2017‐043, PRSTRT Hurricane Relief Funds, and from the U.S. Dept of Education Title‐V‐PPOHA P031M105050 and Title‐V‐Cooperative P031S130068. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, the Puerto Rico Science, Technology and Research Trust or the U.S. Department of Education.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deletion of Arid1a in osteosarcoma enhances aggressive cell phenotypes
Authors: Fatema Kaniz , Ranjit Arina , Barrott Jared , Habashi Ali
Published: June 2021

Osteosarcoma is a form of bone cancer that primarily affects children and young adults. 30 years ago survival rates went from 20% survival to 75% survival with the introduction of aggressive chemotherapy combined with surgery. For the past three decades the survival rate has remained the same despite the increase in targeted therapies to treat other cancers. Our research is aimed toward discovering new drugs that can treat patients that do not respond to traditional chemotherapy. With recent cancer discoveries, osteosarcoma has been identified as a genetically complex disease. In a forward genetic screen using the transposon piggyBac, we discovered a strong correlation between Arid1a gene repression and increased osteosarcoma rates. The ARID1A protein plays a role in epigenetics by directing chromosomes to unwind from histones. In an attempt to discover whether ARID1A contributes to sarcomagenesis, osteosarcoma cell lines were grown in culture dishes. Using CRISPR/Cas9 gene editing, the ARID1A gene was disrupted from the main sequence and various methods were used to test proliferation rates. Not only are we testing gene disruption in cell lines, but we are also testing mouse models that have had Arid1a knocked out to determine if osteosarcoma growth rates increased. Our long term goal is to provide a therapy that can be tested in humans with osteosarcoma to help those who do not respond to traditional chemotherapy and provide an alternative therapy with potentially less side effects.

Support or Funding Information

Idaho INBRE

Idaho State University Start Up

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MacroH2A Variant Suppress Uveal Melanoma Progression and Rewires Cancer Metabolic Phenotype
Authors: Longhitano Lucia , Giallongo Sebastiano , Caltabiano Rosario , Reibaldi Michele , Longo Antonio , Russo Andrea , Fallico Matteo , Tibullo Daniele , Distefano Alfio , Nicolosi Daniela , Lazzarino Giuseppe , Volti Giovanni Li
Published: June 2021

Histone are a family of protein conserved among all the eukaryotes. A histone variant part of the H2A histone family is macroH2A1 which is expressed in nearly all cell types and is involved in a number of processes including cell cycle, gene regulation, DNA damage repair and senescence. Melanoma of the uveal tract, or uveal melanoma (UM), though rare, is one the most common primary cancer of the eye. Its incidence is approximately 4.3 new cases per million people in the United States. Metastasis is a frequent occurrence in uveal melanoma, and outcomes are poor once distant spread occurs and no clinically significant chemotherapeutic protocol is so far available.

The aim of the present study was to evaluate the relationship between macroH2A1 and UM progression. We established a stable knockdown of the protein, using a specific macroH2A1 shRNA and assessed differences in cell proliferation, migration and metabolic profile.

Cell proliferation, migration and colony formation capacity were evaluated by Xcelligence technology, wound healing assay and clonogenic assay. Mitochondrial respiratory chain gene expression was evaluated by RT‐PCR and confirmed by immunocytochemistry whereas metabolic phenotype was determined by Seahorse technology. Our results were further confirmed in a series of human UM biopsies obtained and results were correlated to survival, metastatic outbreak and proliferative index. The study was conducted according to the basic principles of the declaration oh Helsinki. Our results showed that knockdown for macroH2A1 resulted in a reduced proliferation, migration and colony capacity formation rate when compared to their control. Using seahorse assay we also demonstrated that the knockdown of macroH2A1 induced a shift in cell metabolism. These results were confirmed by RT‐PCR and immunocytochemistry showing that knockdown for macroH2A1 had a significant decreased expression of genes implicated in oxidative phosphorylation and in NAD+ metabolism, and a decreased level in TFAM expression. Finally, these data were confirmed in UM biopsies, exhibiting increased expression of macroH2A1 inpatients deceased for metastasis. Furthermore, p16, a well‐established marker of cell proliferation, correlated with macroH2A1 expression. In conclusion, our results showed that macroH2A1 play a major role in UM progression and may represent a possible target for future chemotherapic strategies.

Support or Funding Information

This work was supported by FIR2016/2018 from the University of Catania

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 effect of experience with phonological sequences on the suppression effect, used to assess echoic sensory memory, within the auditory‐evoked potentials (AEPs)
Authors: Kalontar Enessa , Hervey Olivia , Wagner Monica
Published: June 2021

Specific Language Impairment (SLI), a developmental language disorder, has been associated with deficits in working memory (Gathercole, 2006). Using magnetoencephalography (MEG), Helenius and colleagues (2009, 2014) have shown that adults and children with SLI have impaired sensory memory for the repetition of meaningful spoken words. Also, of interest is whether individuals with SLI show impaired sensory memory for phonological sequences, which would likely affect vocabulary learning. As a first step in pursuing this question, we examined the effects of experience with phonological sequences on the suppression effect used to assess sensory memory. The suppression effect, is a reduced response within the auditory evoked potentials (AEPs) or its magnetic counterpart, to repetition of an auditory event, including phonological sequences. EEGs were recorded from 24 native‐English and 24 native‐Polish adults as they listened to same and different non‐word pairs within two experimental sessions. Non‐word pairs contained a phonological sequence contrast, /st‐sət/that occurs in the English and Polish languages and a contrast /pt‐pət/that occurs only in the Polish language. The EEG was time locked to the second word in the non‐word pairs, which was the same non‐word in the same pairs (pətilɑ‐pətilɑ) and different pairs (ptilɑ‐pətilɑ). Thus, a larger AEP response to the second word in the different pairs relative to the second word in the same pairs indicates that the brain registered the additional sound in the different pair. Likewise, evidence of the suppression effect within the same pairs suggest an intact sensory memory for the phonological sequences within the first non‐word. Preliminary examination of results at averaged fronto‐central electrode sites within the AEP revealed an effect of language experience between ~400–600 ms post‐word onset. The native‐Polish listeners showed a larger response to their native language /pt‐pət/contrast than the English listeners and the English listeners showed a larger response to the /st‐sət/contrast that is more prominent in the English language than the Polish language. Thus, native‐language experience and frequency of phonological sequences within a language should be considered when assessing sensory memory in clinical populations.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deletion of Arginase 2 reduces neurodegeneration in a model of Multiple Sclerosis
Authors: Palani Chithra D. , Liu Fang , Xu Zhimin , Fouda Abdelrahman , Caldwell Ruth B. , Narayanan S. Priya
Published: June 2021

Purpose

Visual impairment due to optic neuritis is a major clinical feature of Multiple Sclerosis (MS), and can lead to temporary or permanent vision loss. Previous studies from our lab have demonstrated the critical involvement of arginase 2 (A2) in retinal neurodegeneration. The current study was undertaken to investigate the role of A2 signaling in MS mediated retinal neuronal damage.

Method

Experimental Autoimmune Encephalomyelitis (EAE) was induced in WT mice and A2 knock out (A2 KO) mice (both on C57Bl6 background) according to established procedures. Briefly, WT and A2 KO mice were immunized by subcutaneous injections of an emulsion containing MOG35‐55 peptide (300 μg/mouse) along with Complete Freund's adjuvant (CFA, killed Mycobacterium tuberculosis H37Ra, 400 μg/ul). Each mouse additionally received 300 ng of pertussis toxin (PT, Sigma, St. Louis, MO) by i.p. injection in 200 μl of PBS on day 0 and day 2 post‐immunization. Control group will be immunized with CFA without antigen (MOG), and two doses of PT will be given on day 0 and 2. Clinical disease scores were monitored daily in all groups in a blinded fashion by measuring paralysis according to the conventional grading system. Neurodegeneration studies were performed using retinal flatmount analysis. Expression of arginase and the downstream signaling molecules were examined by Western blotting, and inflammatory molecules were analyzed by qPCR.

Results

Increased expression A2 was observed in WT retinas in response to EAE induction. EAE‐induced motor deficits and body weight loss were markedly reduced in A2 KO mice compared to WT controls. Retinal flat mount studies demonstrated a significant reduction in the number of Brn3a positive and NeuN positive neurons in WT EAE retina (p<0.01; N=6–10) in comparison to normal control mice. A significant increase in the number of surviving neurons (NeuN‐positive and Brn3a‐positive) was evident in retinas of EAE‐induced A2KO mice compared to WT (p<0.05; N=6–10). RNA levels of proinflammatory molecules such as CCL2 ((C‐C motif) ligand 2), COX2 (Cyclooxygenase‐2), IL1a (Interleukin 1a), IL12a, and IL‐18 were significantly reduced in the A2KO EAE retinas compared to WT EAE (p<0.05, N=5–6). Signaling studies showed increased levels of phospho‐ERK1/2 and reduced levels phospho‐BAD in the WT EAE retina, while these changes were normalized in EAE‐induced A2 retinas.

Conclusion

Our studies establish EAE as an excellent model to study MS‐mediated retinal neuronal damage and suggest the potential value of targeting arginase 2 signaling as a therapy to prevent MS‐mediated vision loss.

Support or Funding Information

These studies supported by the National Multiple Sclerosis Society (PP‐1606‐08778 to S.P.N) and Augusta University Culver Vision Discovery Institute.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Co‐localization of Glia Maturation Factor and Progranulin in the Human Alzheimer's Disease Brains
Authors: Thangavel Ramasamy , Premkumar Keerthivass , Zahoor Haris , Saeed Daniyal , Iuliia Dubova , Pushpavathi Selvkumar Govindhasamy , Duraisamy Kempuraj , Ahmed Mohammad Ejaz , Raikwar Sudhanshu P , Zaheer Smita A , Iyer Shankar S , Zaheer Asgar
Published: June 2021

Amyloid plaques (APs) and neurofibrillary tangles (NFTs) are two major neuropathological hallmarks observed in the brains of Alzheimer's disease (AD) patients. Inflammatory mediators released from the activated glial cells and neurons induce neuroinflammation and neurodegeneration thereby exacerbating AD pathogenesis. We have previously shown that glia maturation factor (GMF), a proinflammatory brain protein is upregulated in the brains of AD patients. Additionally, we also found that the upregulated GMF is associated with APs and NFTs in the brains of AD patients. Progranulin a pleiotropic protein that serves as a precursor of active granulin peptides was also shown to be upregulated in the brains of AD patients. However, the association between GMF and progranulin has not yet been studied in the context of neurodegenerative diseases including AD. We analyzed frozen sections of temporal lobe from human AD patients in this study. Here, we investigated whether the upregulated GMF expression is associated with a corresponding increase in progranulin expression in the vicinity of APs in the AD brains using single and double immunohistochemistry and immunofluorescence staining. Our results show that progranulin immunoreactive neurons and activated microglia are present in the temporal cortex region of AD brains. We report increased progranulin and GMF expression in the vicinity of APs. Further, we also report significantly more progranulin expression in the neurons and glial cells in the brains of AD patients. Our data suggest that the increased expression of GMF and progranulin may synergize to play a critical role in neuroinflammation and neurodegeneration thereby exacerbating AD pathogenesis.

Support or Funding Information

This work was supported by the Veterans' Affairs Merit Award I01BX002477 and the National Institutes of Health Grants AG048205 and NS073670 to AZ

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Eliciting Differentiation of Human T Lymphocytes into TH1 or TH2 cells with Inorganic Arsenic Exposure
Authors: Jewell Mollie Anne , Nyland Jennifer
Published: June 2021

A naturally‐occurring element, inorganic arsenic is found around the world in soil. It can contaminate fresh groundwater, where it is at risk of being ingested. Currently, arsenic is considered to be dangerous with chronic exposure and ingestion at 10 ug/L as established by the World Health Organization and the U.S. Environmental Protection Agency. Chronic exposure to arsenic is associated with inflammation, but current studies have show that even at lower doses, there is a link to expression and secretion changes of cytokines within T lymphocytes, particularly with IL‐2 for growth, pro‐inflammatory IFN‐γ and anti‐inflammatory IL‐4. Shifts favoring pro‐ or anti‐inflammatory cytokines can lead to differentiation into TH1 or TH2 cells, which specialize in their immune response. In this study, we aim to investigate how the expression and secretion of these cytokines produced by activated T lymphocytes change as an in‐vitro model of an activated human regulatory immune system. Through our model, we will detect if chronic low level arsenic exposure has the potential to catalyze a change in T lymphocyte function, and therefore overall immune function.

Previous studies have utilized various conditions by which to activate and treat T lymphocytes. As a result, there is variation in how these cytokines are predicted to change. For this study, we obtained human T lymphocytes. They were treated over 24 hours with 10 ng/mL of phytohemagglutinin, a known immune activator, and different concentrations of arsenic to develop a dose response curve, from 20 nM to 500 nM. Viable concentration and cytolethality were determined with differential staining and counting. Cell concentration and cytolethality changes were examined with one‐way ANOVA and Bonferroni's Pairwise T‐test. At 50 nM there is a difference in the cytolethality of the cells in the groups treated with arsenic with or without PHA with p < 0.05.

Future work will include ELISA assays will be performed on all treatment groups of the cell media collected for IL‐2, IL‐4, and IFN‐γ, followed by gene expression analysis via PCR. Experiments at 50 nM will be repeated as well to verify differences in cytolethality that were identified. Additionally, differential time periods for treatments of arsenic and PHA will be used to more accurately model chronic exposure in the cells. These other time points will analyzed through ELISA and gene expression analyses to inform the model of long‐term change within the cells of the cytokine profiles.

Support or Funding Information

This research is supported by the Salisbury University Department of Biological Sciences, the Salisbury University Honors College, and the Salisbury University Henson School of Science 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.
The hydrogel‐encapsulated liver X receptor ligand, T0901317, enhances its antitumorigenic effects but eliminates lipogenesis
Authors: Feng Ke , Yang Xiaoxiao , Ma Chuanrui , Chen Yuanli , Han Jihong , Duan Yajun
Published: June 2021

The anti‐tumorigenic functions of liver X receptor (LXR) ligands have been confirmed in several types of tumors. We previously reported that LXR ligand, T0901317 (T317), inhibited growth of inoculated LLC1 carcinoma and urethane‐induced pulmonary carcinomas by activating IFN‐γ expression in tissues, particularly in macrophages. However, T317 activates fatty acid synthesis to result in severe hepatic lipogenesis and fatty liver. Tissue‐specific activation of LXR, such as in antigen‐presenting cells (APCs) but not in liver, by ligand can enhance inhibition of tumor growth without activation of lipogenesis. Injected naphthylacetic acid modified D‐glycine‐phenylalanine‐phenylalanine‐tyrosine tetra‐peptide (GFFY) can be selectively delivered to APCs, macrophages and dendritic cells (DCs). In this study, we encapsulated T317 with GFFY hydrogel (GFFY‐T317) and determined its effect on tumor growth and hepatic lipogenesis. Compared with oral administration of T317, subcutaneous injection of GFFY‐T317 more potently inhibited LLC1 tumor growth in mice. The inhibition was dependent on induction of IFN‐γ expression in APCs by activating LXR. We further determined that GFFY‐T317 increased M1 type macrophages while reducing M2 cells in the tumor. Meanwhile, GFFY‐T317 enhanced DC maturation and infiltration to tumors. Both CD3+ and CD8+ cells in tumors were also increased, which was associated with activation of IFN‐γ expression. In contrast, tumor angiogenesis was clearly inhibited by GFFY‐T317 in an IFN‐γ‐dependent manner. In addition, GFFY‐T317 had more potent inhibitory effects on urethane‐induced lung tumor than oral administration of T317. More importantly, in these two tumor models, compared with severe liver steatosis by oral administration of T317, injection of GFFY‐T317 had little effect on lipid synthesis/metabolism in the liver, while eliminating T317‐induced liver injury. Taken together, our study demonstrates that the hydrogel‐encapsulated T317 can inhibit lung tumor growth without adverse effect of lipogenesis, indicating the hydrogel‐encapsulated LXR ligand might be a novel therapy for tumor 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.
Angiotensin‐converting enzyme (ACE) up‐regulates metabolic function in myeloid‐derived cells
Authors: Cao DuoYao , Khan Zakir , Peng ZhenZi , Giani Jorge F , Veiras Luciana C , Chang Corey , Bernstein Ellen , Bernstein Kenneth
Published: June 2021

While most studies of ACE focus on the blood pressure effects of angiotensin II, it is now recognized that ACE also plays an important role in both the innate and adaptive immune responses1. To study the mechanism of this effect, we made two mouse strains: ACE 10/10, which over‐express ACE in macrophages, and NeuACE, which over‐express ACE in neutrophils. NeuACE and ACE 10/10 mice have a better immune response in several different disease models (e.g. bacteria, tumor, Alzheimer's disease)1,2. We hypothesized that ACE affects myeloid function by inducing changes in cellular energy metabolism. To examine this, metabolic intermediates were studied by mass spectrometry and chemical methods in ACE over‐expressing cells.

Methods

Neutrophils were isolated from bone marrow; macrophages were collected from the peritoneum 4 days after thioglycolate injection. Cells were purified by Percoll gradient.

Results

Mass spec. showed more than 32 differences, with virtually all affected metabolites increased in the ACE over expressing cells. In ACE 10/10 macrophages, cellular ATP, ADP and AMP was 3.4‐, 2.7‐, and 2.7‐fold the levels in WT cells. In NeuACE neutrophils, the pattern was the same but numerically less than in macrophages. Chemical analysis showed a 2.8‐fold and a 1.5‐fold increase of cellular ATP in ACE 10/10 macrophages and NeuACE neutrophils. In ACE 10/10 macrophages, the ATP/ADP ratio was 2.1 vs. 1.4 for WT cells. For NeuACE neutrophils, the ATP/ADP ratio was 3.0 vs. 1.9 in WT neutrophils. The increase of cellular ATP is due to the catalytic activity of ACE; when animals were treated with an ACE inhibitor, peritoneal macrophages or bone marrow derived neutrophils showed ATP levels that were no different from similarly treated WT mice. The effect of ACE is not due to increased angiotensin II.

Conclusions

ACE overexpression in macrophages or neutrophils of mice upregulates ATP concentrations in these immune cells. This is due to the catalytic activity of ACE but not the production of angiotensin II. Increased cellular ATP plays an important role in the enhanced innate and adaptive immune ability of myeloid cells over expressing ACE.

Support or Funding Information

NIH P01 HL129941 and R01 AI143599

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This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
S‐sulfhydration of SIRT3 by hydrogen sulfide attenuates mitochondrial dysfunction in cisplatin‐induced acute kidney injury
Authors: Yuan Yujia , Li Lan , Zhu Lingling , Chen Younan , Liu Jingping , Lu Yanrong , Cheng Jingqiu
Published: June 2021

Clinical use of cisplatin, one of the most widely used common and effective chemotherapeutic agents, is limited by its side effects, particularly tubular injury–associated nephrotoxicity. Mitochondrial dysfunction is one of the main mechanisms contributing to cisplatin‐induced acute kidney injury (AKI), and sirtuin 3 (SIRT3) is closely associated with mitochondrial function and oxidative stress. Hydrogen sulfide (H2S) has powerful anti‐oxidative and anti‐inflammatory properties. We report that H2S alleviated cisplatin‐caused renal toxicity via SIRT3 activation and subsequent mitochondrial bioenergetics. Using a biotin switch assay, we show that H2S increased S‐sulfhydration of SIRT3 and induced deacetylation of its target proteins (OPA1, ATP synthase β and SOD2). These effects of H2S were associated with a reduction of mitochondrial fragment, an increase in ATP generation, and less oxidative injury. Notably, the S‐sulfhydration of SIRT3 induced by NaHS (a H2S donor) was abrogated when Cys256, Cys259, Cys280, and Cys283residues on SIRT3 (2 zinc finger domains) were mutated. Collectively, we demonstrate that H2S attenuates cisplatin‐induced AKI by preventing mitochondrial dysfunction via SIRT3 sulfhydrylation. Thus, SIRT3 may represent a new therapeutic target and H2S may be a potentially effective agent to limit cisplatin‐induced nephrotoxicity by activating SIRT3.

Support or Funding Information

This work was supported by the National Natural Science Foundation of China (Grant number 81370824 and 31571474), and China Postdoctoral Science Foundation (2017M620428).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Facilitating fructose‐driven metabolisms increases a capability to resist anoxic stress in Drosophila
Authors: Kim Man Su , Kim Hongjo , Kim Misun
Published: June 2021

Hypoxic stress is linked to various cardiovascular disorders including stroke and myocardial infarction. A key pathophysiological process upon hypoxic stress is a reduction in ATP generation. However, a fructose‐driven glycolysis has been proposed as an alternative biochemical pathway, which could sustain ATP production even in anoxic stress. Here we tested the hypothesis that facilitating fructose‐driven ATP generation increases organisms' capability to withstand anoxic stress using a Drosophila as an in vivo animal model. Genetically modified flies that contain human fructose transporter (GluT5) and ketohexokinase (KHK) sequence downstream of UAS were constructed. Using a GAL4‐UAS system that allows us to express GluT5 and KHK in a tissue‐ and time‐dependent manner, it was found that overexpression of both genes in whole flies (with Act5c‐gene switch GAL4 driver) or in neurons (with elav‐gene switch GAL4 driver) reduces flies' mortality upon anoxic stress. Besides, it was found that spontaneous locomotor activity of the GluT5/KHK overexpression flies was higher than that of control flies upon anoxic assaults. Taken together, these data suggest that enhancing fructose metabolisms could be utilized as a potentially clinically relevant approach to minimize hypoxia‐induced cellular damages.

Support or Funding Information

This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and Technology (NRF‐2017R1E1A2A01079316)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Role of the Escherichia coli Lysophospholipase PldB in Determining Cell Length
Authors: Nikpour Naveed , Garrett Teresa A
Published: June 2021

Headgroup acylated glycerophospholipids (GPLs) are proposed to carry out an essential function in cell division in the cell membranes of Escherichia coli. Lysophospholipase L2 (PldB) has been shown to transfer the acyl group from 2‐acyl‐lysophospholipids to the headgroup of phosphatidylglycerol (PG) to form acyl PG. Cells overexpressing PldB have been found to show an elongated cell phenotype. As the cause for this phenotype is not entirely known, we aim to develop a robust assay to test PldB activity so as to understand what aspect of PldB activity is implicated in the cell elongation phenotype. We are developing a radiochemical thin‐layer chromatography assay for PldB using 14C PG and 14C (bis‐monoacylglycerol)phosphate to the study this mechanism. Using liquid chromatography‐mass spectrometry‐based assays, we are testing cells with mutations to PldB's catalytic triad to determine a catalytically dead mutant. This mutant would allow us to assess if cell elongation requires PldB activity or if PldB's association with the inner membrane impacts the activity of cellular division machinery. This project aims to undercover more information about the functioning of PldB and headgroup acylated GPLs. Due to PldB's role in cell membrane integrity, further analysis of its mechanism may further our understanding of how the cell membrane functions during cell division. Future research can focus on localizing PldB within the membrane to help assess PldB's function during cell division.

Support or Funding Information

This work was funded by National Science Foundation Research at Undergraduate Institution Award #1516805 to T. A. G.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhancement of Lipid Signal in MALDI MS imaging obtained from Formalin Fixed Human Brain Tissue
Authors: Harris Aaron , Mor Rahul , Whitehead Shawn , Yeung Ken
Published: June 2021

Matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is used to perform mass spectrometric analysis directly on biological samples providing accurate visual/anatomical spatial information of molecules within the tissue. A current limitation of MALDI‐IMS is that it is largely performed on fresh frozen tissue whereas clinical tissue samples stored long term are fixed in formalin. It has been shown that fresh frozen tissue sections applied with an ammonium formate (AF) wash prior to matrix application in the MALDI‐IMS procedure display an increase in observed signal intensity and sensitivity for lipid molecules detected in the brain while maintaining the special distribution of molecules throughout the tissue. In this work we investigate the effectiveness of this AF wash on post‐fixed rat and human brain tissue sections in an effort to increase the viability of formalin fixed tissue imaging in a clinical setting. Results herein demonstrate that the AF wash significantly improved MALDI‐IMS spectra for gangliosides, including GM1 in fresh frozen rat brain, formalin‐fixed rat brain and formalin fixed human brain samples. AF wash also demonstrated improvements in MALDI‐IMS image quality while retaining the spatial distribution of molecules. Results indicate that this method will allow analysis of gangliosides from formalin‐fixed clinical samples, which can open additional avenues for neurodegenerative disease 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.
Investigating the Kinetics of the Arabidopsis thaliana Acyltransferase At1g78690
Authors: Jahns Max , Garrett Teresa A
Published: June 2021

Headgroup acylated glycerophospholipids (GPLs) are a class of membrane phospholipids containing an acyl‐chain attached to the headgroup of the molecule that make up a relatively small portion of the cell envelope but are implicated in important functions such as the cell stress response and cell division. At1g78690 is an acyl‐transferase native to Arabidopsis thaliana that converts mono‐acylated GPLs to diacylated GPLs in vitro. Interestingly, overexpression of At1g78690 in E. coli leads to an accumulation of the headgroup acylated GPL acyl‐phosphatidylglycerol (acyl‐PG) in vivo. While At1g78690 is known to be capable of transferring an acyl chain from acyl‐CoA to two isoforms of bis(monoacyl glycerol) phosphate (BMP), 3,1’ BMP and 3,3’ BMP, to form acyl‐PG, it does not acylate 1,1’ BMP suggesting the enzyme possesses some stereoselectivity that needs to be further investigated. We reevaluate the extent to which At1g78690 is substrate selective, by further characterizing the ability of At1g78690 to acylate PG and 1,1 BMP. We also estimate the Km of At1g78690 on lyso‐PE, lyso‐PG, lyso PS, lyso‐PI, and 3,1’ and 3,3’ BMP. The kinetics of At1g78690 are measured via an acyl‐chain acceptor (either lyso‐PG, lyso‐PS, lyso‐PI, 3,1’ BMP or 3,3’ BMP) receiving a 13C‐labeled acyl chain from acyl‐CoA. The kinetics of an enzyme can give insights into its substrate specificity, its catalytic and energetic properties, and its role in cellular phospholipid synthesis.

Support or Funding Information

This work was funded by National Science Foundation Research at Undergraduate Institution Award #1516805 to T. A. G.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Gylcerophosphocholine Acyltransferase Homolog, Gpc1, in Candida albicans
Authors: King William Ray , Patton‐Vogt Jana
Published: June 2021

The maintenance of membrane composition through regulated synthesis and turnover of phospholipids is crucial to cellular homeostasis. Phosphatidylcholine (PC) is the most abundant phospholipid in most eukaryotic membranes. We have identified a glycerophosphosphocholine acyltransferase (GPCAT), Gpc1, that expands our understanding of PC biosynthesis and acyl chain remodeling in Saccharomyces cerevisiae. Gpc1 acylates glycerophosphocholine (GPC), a product of complete deacylation of PC, to form lysophosphotidylcholine (LysoPC). LysoPC can subsequently be converted to PC by Ale1. This PC deacylation reacylation pathway (PC‐DRP) defines a novel route for PC biosynthesis and provides a potential mechanism to alter the acyl chain composition of a PC molecule. Changes in acyl chain composition can, in turn, affect physical properties of a membrane, such as fluidity, width, thickness and shape. Gpc1 displays no homology to known acyltransferases and defines a new gene family with members in animals, plants and fungi. The pathogenic fungus Candida albicans displays robust transport of GPC into the cell. We have previously shown that the inability of C. albicans to transport GPC results in decreased virulence in a mouse model of disseminated candidiasis implying this metabolite may play an important role during pathogenicity. Here we report on the creation of a C. albicans diploid knockout of Gpc1 homolog (orf19.998) using a transient CRISPR Cas‐9 system. Radiolabeling coupled with mass spectrometry are being employed to determine if the caGpc1 is similar to the scGpc1 in terms of biochemical function. Initial studies to examine the physiological role of caGpc1 are being performed.

Support or Funding Information

NIH R15GM104876 (to JPV)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Membrane Microdomains on a Human Granulosa Cell Line by Atomic Force Microscopy
Authors: Brogie Molly , Cohen Brian D
Published: June 2021

Signaling via the Hypothalamic‐Pituitary‐Gonadal (HPG) axis regulates fertility in both men and women. Follicle stimulating hormone (FSH), secreted from the anterior pituitary, binds to a G protein coupled receptor (GPCR) on ovarian granulosa cells. The activation of this receptor triggers a cellular signaling pathway that leads to the promotion of follicle development and estrogen secretion. Our lab has previously demonstrated that the human FSH receptor (hFSHR) shows an affinity for residing in lipid rafts of HEK293 cells which may play a pivotal role in its signaling. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids, with higher stiffnesses than bulk membrane domains. To characterize lipid rafts on the human granulosa cell line hGrC1 we used atomic force microscopy. Atomic force microscopy is a technique used to produce nanoscale resolution topographical images. It can also be used for determining mechanical characteristics, such as stiffness and strain. Here, using contact and force‐volume mode atomic force microscopy, we successfully generated topographical maps of hGrC1s and recorded membrane stiffness measurements. Areas of the granulosa cell membrane showed variable stiffness on a scale of 58.998 nm / pixel, providing the first direct evidence of lipid rafts in human granulosa cell membranes. Demonstration of lipid rafts contributes to the implications of the role of lipid rafts hFSH signalling. By understanding membrane dynamics on granulosa cells we hope to gain insight into how to manipulate hFSHR 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.
DPMS is Essential for Tumor Angiogenesis
Authors: Collazo‐Irizarry Diego , Pabon Andrea Firpo , Ramirez‐Alameda Ricardo , Baksi Krishna , Banerjee Dipak K.
Published: June 2021

Dolichol phosphate mannose synthase (DPMS) an inverting GT‐A folded enzyme and classified as GT2 by CAZy (carbohydrate active enZyme; http://www.cazy.org). It catalyzes the transfer reaction Dol‐P + GDP‐Man < == > Dol‐P‐Man + GDP in endoplasmic reticulum (ER) for the synthesis of dolichol‐linked tetra‐decasaccharide [Glc3Man9GlcNAc2‐PP‐Dol; lipid linked oligosaccharide (LLO)], a pre‐requisite for the glycosylation of asparagine‐linked (N‐linked) glycoproteins. N‐Glycosylation provides structural and functional stability, and adds flexibility to glycoproteins. The gene for DPMS has been cloned from many species, i.e., from archaea to human. The amino acid sequence identifies the presence of a metal binding (i.e., DAD) signature and a cAMP‐dependent protein phosphorylation (PKA) motif. Phosphorylation enhances the DPMS catalytic activity and consequently increases the synthesis and turnover of LLO. In capillary endothelial cells, it means increased proliferation, i.e., angiogenesis. Angiogenesis is a hallmark for breast cancer progression. Therefore, we have hypothesized that overexpression of DPMS in capillary endothelial cells could mimic the cAMP signaling and independently increase angiogenesis. To test the hypothesis, we have used a cell line that stably overexpresses DPMS and evaluated the role of D(+)mannose on their proliferation by MTT assay. Capillary endothelial cells harboring the DPMS overexpressing plasmid exhibited increased expression of DPMS when examined by immunofluorescence microscopy, western blotting, and QPCR. There was increased expression of cell surface N‐glycans as judged by Texas‐Red conjugated WGA‐binding. The cell proliferation as well as the cellular mitotic activity were also enhanced. To evaluate if mannose is required for the observed effect, we have monitored the cellular proliferation as a function of mannose concentration and the time of incubation by MTT assay. The results indicate mannose supplementation did not support the growth of neither the DPMS overexpressing cells nor the control cells. Triple negative human breast cancer cells also showed a similar result. We therefore conclude DPMS overexpression induces angiogenesis. Mechanistically it is not due to upregulation of mannose transport. The environmental factors increasing intracellular cAMP level increase DPMS catalytic activity metabolically or through gene expression and enhance angiogenesis for breast tumor progression. DPMS, thus could be an excellent target for developing a new generation glycotherapy against breast cancer.

Support or Funding Information

Institutional

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of an antifungal peptide, Diapausin, from Manduca sexta
Authors: Li Miao , Al Souhail Qasim , Veerapandian Raja , Vediyappan Govindsamy , Kanost Michael
Published: June 2021

Antimicrobial peptides, a first line of defenses against the infectious microbes, are present in bacterial, fungal, plant and animal kingdoms. They are typically cationic and are less than 50 amino acid residues long. Most known insect antimicrobial peptides are bactericidal, and a few target both bacteria and fungi with relatively broad spectrum, but only a small group is specifically active against fungi. Manduca sexta, the tobacco hornworm, is an established model for biochemical studies of insect immunity. Eighty‐six antimicrobial peptide genes from 12 families were identified in the M. sexta genome. We recently discovered diapausins as a family of antifungal peptides in M. sexta hemolymph, active against Saccharomyces cerevisiae and some ascomycete fungi. Diapausin concentration increases in hemolymph after injecting the larvae with yeast or bacteria. Purified diapausin‐1 and ‐2 recombinant proteins expressed in E. coli block growth of S. cerevisiae with IC50 of 30uM and 60uM, respectively. Diapausin‐1 and ‐2 disrupt daughter cell separation after cell division, resulting in cell clusters. Diapausin‐1 is active against the human fungal pathogen Candida genus as well. The growth of Candida albicans and Candida krusei is inhibited by diapausin‐1, with IC50 of 60uM and 20uM respectively, and diapausin‐1 treatment alters the morphology of these fungi. Diapausin‐1 impairs the germination of spores and hyphal growth of Beauveria bassiana, an insect pathogenic fungus. FITC‐labeled diapausin‐1 binds to the surface of S. cerevisiae, and pull‐down experiments show that diapausin‐1 binds to □‐1,3‐glucan, a component of fungal cell walls. We are investigating the mechanisms of diapausin action using □‐1,3‐glucan synthase (FKS1) mutants from S. cerevisiae and C. albicans. Diapausin‐1 may disrupt fungal growth by binding to □‐1,3‐glucan and/or impairing □‐1,3‐glucan synthesis. This study advances our understanding of an insect immune response to fungal infections and may contribute to the identification of new targets for the development of antifungal drugs.

Support or Funding Information

NIH grants GM041247, Kansas State University Division of Biology's startup funds, AHA grants 4SDG18910036

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Microstructure Detection by Scanning Acoustic Microscope (SAM) using Special and Antigen Staining
Authors: Miura Katsutoshi , Fujie Michio , Yamashita Kanna
Published: June 2021

Objective

A SAM detects and visualizes cellular components and extracellular matrix (ECM) proteins. However, which materials contribute to the values of speed‐of‐sound (SOS) or attenuation‐of‐sound (AOS) is unknown. SOS and AOS indicate tissue stiffness or softness because greater SOS and AOS areas correspond to stiffer portions of tissues. The aim of this study is to clarify the role of each component in SAM observation.

Methods

To detect each component, special staining for ECM and cellular components such as collagen, elastin and DNA were used. To recognize specific antigens, immunostaining with Ni‐DAB solution as horseradish peroxidase substrate was applied. SAM images before and after staining were compared.

Results

For nuclear detection, both hematoxylin and Giemsa staining resulted in increased SOS and AOS values of the nuclei (Fig. 1 and Fig. 2). To distinguish ECM components, aniline blue (Fig. 3) and Sirius red for collagen and resorcin‐fuchsin (Fig. 4) for elastin stained well to increase SOS and AOS values according to duration of incubation. After collagenase digestion, SOS and AOS values at the positive‐staining areas decreased. Immunostaining with Ni‐DAB solution raised SOS and AOS values of antigen location (Fig. 5).

Conclusions

Special and immunostaining used in light microscopy are also available to certain the antigen location for SAM. Collagens and smooth muscles contribute to raise the values of SOS and AOS.

Support or Funding Information

The study was supported by a grant from the Japan Society for the Promotion of Science KAKENHI, Scientific Research Grant Number (c) 15K08375. There are no conflicts of interest to declare.

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