bluuebunny/crossref_metadata_2025 huggingface dataset:
Computational Insights into the Structure and Epitope locations for Two Idiopathic Membranous Nephropathy Antigens: Phospholipase A2 Receptor and the Thrombospondin Type‐1 Containing Domain 7A and Design of Antigen Binding Proteins
Authors: Stoddard Shana
Published: June 2021

The use of non‐specific treatment options in autoimmune therapy today unfortunately causes patients to be prone to other infections. This non‐specific therapy suppresses the entire patient's immunes system also making it harder to fight of basic infections. Thus therapies need to be developed which can accurately target only the aberrant portion of the immune system that contributes to the autoimmune disorder (AD) specific to the individual. In the AD idiopathic membranous nephropathy (IMN) two antigens have been determined to produce an immune response. The work here focuses was performed to aid in the design of antigen specific binding proteins (ABP) as a targeted means to treat AD, and specifically IMN. In this work a homology model of the thrombospondin type‐1 containing domain 7A (THSD7A), was successfully developed, and epitope regions were predicted using Epitopia. In silico mutagenesis was utilized to develop ABP for both THSD7A and phospholipase A2 receptor (PLA2R). Using Rosetta protein‐protein docking simulations evaluate for maximum binding efficiency against the ricin and the CTLD2 epitopes present on theTHSD7A and PLA2R antigens. Our results show that THSD7A has 22 domains and 12 domains have potential to contain epitope sites. It was determined that there were two possible epitope locations in CTLD1 domain and three epitope locations in CTLD7 domain of the PLA2R antigen. Our results show improved binding scores and selectivity of ABP against the PLA2R. Initial binding energy, measured in rosetta energy unit (REU), were −2.863 and −2.633 for the SH13 template and ySMB‐1 ABP templates respectively against the PLA2R antigen. The M005 mutation designed of the SH13 improved binding scores to −5.32 REU. Mutations to the ySMB‐1 template were shown to be able to enhance scores to −6.907 REU. Molecular interactions leading to enhanced potency have also being evaluated. ABP candidates for the THSD7A antigen have also been designed. A total of fifteen ABP candidates have been selected for expression which was performed in E. coli followed by subsequent purification using affinity chromatography for experimental verification of computational results is using ELISA. This work has implications for new antigen specific approach to AD therapy.

Support or Funding Information

Faculty Development Endowment Grant, Rhodes College

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cytotoxic Mechanism of Long‐chain Lipids Extracted from Mexican Native Avocado Seed (Persea americana var. drymifolia) on Colon Cancer Cells
Authors: Lara‐Márquez Mónica , Spagnuolo Paul A. , Salgado‐Garciglia Rafael , Ochoa‐Zarzosa Alejandra , López‐Meza Joel Edmundo
Published: June 2021

Cancer represents a worldwide problem, in 2012 about 8.2 million deaths were reported. Colorectal cancer (CRC) is the third most common type of cancer and the fourth leading cause of death in the world, accounting for about 700,000 deaths a year. Chemotherapy currently used in patients with CRC is associated with severe side effects. The use of natural compound combinations extends the possibilities of therapeutic targets. Long chain lipid molecules, such as long chain fatty acids, aliphatic acetogenins and polyhydroxylated fatty alcohols independently have been shown to have anticancer effects and have been proposed as chemo‐therapeutic adjuvants. The main objective of this work was to determine the cytotoxic effect of an extract rich in long chain lipids from Mexican native avocado seed (Persea americana var. drymifolia) on colon cancer cells. Colon adenocarcinoma cell line Caco‐2 was used and viability was measured by trypan blue exclusion assays. Changes in mitochondrial membrane potential, cytokine expression, intracellular calcium levels, cell cycle analysis and ROS production were determined by flow cytometry. The induction of apoptosis was analyzed by flow cytometry; spectrometry and fatty acid oxidation (FAO) rate determination was made using high resolution respirometry. The results showed that the long chain lipids from avocado seed induced apoptosis and the activation of the caspases 8 and 9, loss of the membrane mitochondrial potential (68%), the inhibition of FAO (45%) and the increase in ROS production (30%); however, the intracellular calcium levels were not modified. In addition, cell cycle arrest in the G1/G0 phase was induced. Also, a modulation in cytokine expression (IL‐6, IL‐8, IL‐10 and IL‐1β) was observed. In conclusion, long‐chain lipids from Mexican native avocado seed are cytotoxic towards Caco‐2 cells, inhibiting the FAO which increases ROS production and thus favoring the loss of membrane mitochondrial potential that allows the induction of caspase‐dependent apoptosis. In addition, these molecules induce arrest of the cell cycle and the modulation of cytokines related to the inflammatory response.

Support or Funding Information

CIC‐UMSNH 14.5

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluating the Inhibition of Palmitoylation and NHE1 on Cell Proliferation and Migration
Authors: Hanowski Stephanie A. , Provost Joseph J. , Wallert Mark
Published: June 2021

The Na+‐ H+ Exchanger Isoform I (NHE1) is a key regulator of cell proliferation and migration in a range of cell types from both healthy and diseased tissues. Activation of NHE1 is an initial step in the development of the transformed phenotypes of cancer cells and therefore a key driver for tumor development and cancer progression. NHE1 also contributes to two of the hallmarks of cancer, the activation of invasion and metastasis and sustained proliferative growth. We have identified for the first time, that NHE1 undergoes reversible palmitoylation, the addition of a palmitic acid residue to the cytoplasmic regulatory domain of NHE1. We hypothesize that this process regulates NHE1 activation and will play a role in the control of cell proliferation and migration. This suggests that targeting the potential palmitoylation sites on NHE1 may provide a therapeutic target for cancer treatment. Our work characterizes the role of the palmitoylation of NHE1 in cell growth and migration. Using the palmitoylation inhibitor, 2‐bromopalmitate (2BP), we evaluated cell growth and migration in the presence and absence of palmitoylation. In proliferation assays, the rate of cell growth increased approximately 5‐fold when the serum present in the growth media was increased from 0.5% to 10% and approximately 2‐fold when 50 μM lysophosphatidic acid was added to the growth media. Under both conditions, the addition of 15 μM 2BP or 10 μM cariporide dramatically reduced or eliminated the increased proliferation. Cell migration was evaluated using Electric Cell‐substrate Impedance Sensing (ECIS). Once again, the rate of cell migration was reduced by 50% or more in the presence of 15 μM 2BP or 10 μM cariporide. These data demonstrated that inhibiting the palmitoylation of NHE1 decreases both cell proliferation and the rate of cell migration.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effect of Resveratrol in the Proliferation and Autophagy of Breast Cancer Cell Lines
Authors: Casillas Maryangie Martinez , Forti Kevin Munoz , Rivera Jose Robles , Torres Geraline Trossi , Martinez Edu Suarez , Rivera Abigail Ruiz
Published: June 2021

Breast cancer (BC) is one of the most universal malignant tumors of worldwide in women and it is a significant public health problem in terms of both morbidity and mortality. BC is the second leading cause of death among American women. It is estimated 250,000 new cases of invasive BC will be diagnosed in women in 2017. For instance, there still existing the need for developing or identify new effective compounds to fight this disease. For example resveratrol, which is a plant derived polyphenol with anti‐estrogenic and antioxidant properties. In‐vitro studies indicated resveratrol has anticancer properties such as cell cycle arrest, apoptosis induction, prevention of DNA damage, and angiogenesis inhibition. However, scientific literature studying the effect of resveratrol on DNA synthesis and in autophagy is limited. For this reason we studied the effect of resveratrol in the proliferation of three breast cell lines CCD‐1074 (non‐malignant cells), CCD‐1097 (transitional cells), and MCF‐7 (malignant non‐invasive cells); and autophagy induction in the MCF‐7 cell line. We hypothesized that resveratrol will decrease cell proliferation and increase autophagy in BC cells when compared to control treatments. We studied the proliferation effect in cells treated with resveratrol at 25, 50, 100, 200 μM during 24 hours post treatment. We used Bromodeoxyuridine (BrdU) and the detection of translocation of LC3 via flow cytometry to study proliferation and autophagy, respectively. Our results showed a significant decrease in cell proliferation at resveratrol concentrations of 100 and 200 μM across all cell lines when compared to the vehicle control (p<0.05). Importantly, at 50 μM resveratrol affected only transitional and malignant cell lines without affecting our non‐malignant cell line. Induction of autophagy was significantly higher when MCF‐7 cells were treated with 100 μM and 200 μM compared to the vehicle control (p <0.05). However, we are currently in the process of evaluating the effects of resveratrol in other malignant and non‐malignant cell lines. We consider that the 50 μM concentration as a possible therapeutic window selectively to inhibit cell proliferation and induce autophagy.

Support or Funding Information

Do not apply

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calcitriol increases leptin expression in neuronal cells ‐ Implications for Alzheimer's Disease
Authors: Marwarha Gurdeep , Ghribi Othman
Published: June 2021

Alzheimer's disease (AD) is a devastating progressive neurodegenerative disorder that is neuropathologically characterized by the intracellular accumulation of hyperphosphorylated protein tau as neurofibrillary tangles and the extracellular deposition of amyloid‐beta (Aβ) peptide as amyloid plaques. However, the most accurate pathological hallmark and clinical correlate of cognitive impairment as well as deficits in learning and memory is synaptic loss. Leptin is a pleiotropic adipocytokine known to be expressed endogenously in the mammalian brain that plays an indispensable role in learning and memory. Leptin is a bona‐fide neurotrophic cytokine and a growth factor that is integral in the regulation of a multitude of physiological and biochemical neuronal processes. Emerging evidence has implicated that the dysregulation and attenuation of leptin expression in the brain may lead to the initiation and fostering of deleterious neurodegenerative cascades that could culminate in cognitive dysfunction, learning and memory deficits, as well as AD‐like pathological hallmarks. More importantly, leptin decreases Aβ plaques and hyperphosphorylation of protein tau, two pathological hallmarks of AD, and rescues cognitive impairment in several rodent models of AD. A multitude of epidemiological studies, prospective and cross‐sectional, have implicated that lower plasma vitamin D3 levels are associated with a higher risk for developing AD. Contemporary studies have shown that vitamin D3 can regulate leptin expression in the adipose tissue and modulate peripheral circulating plasma levels of leptin. However, no studies have yet examined the effects of Vitamin D3 on leptin expression in the brain and neuronal cells. In this study, we determined the effects of calcitriol (1,25 dihydroxyvitamin D3) on leptin expression in cultured SH‐SY5Y human neuroblastoma cells. Herein, we show that calcitriol (1,25 dihydroxyvitamin D3) increases leptin expression levels by Vitamin D Receptor (VDR) – dependent and – independent mechanisms. Further elucidation and delineation of the underlying mechanisms unveiled the indispensable involvement of the CCAAT/Enhancer Binding Protein alpha (C/EBPα) in the 1,25 dihydroxyvitamin D3 (calcitriol) – induced increase in leptin expression. Our study is the first to demonstrate that 1,25 dihydroxyvitamin D3 (calcitriol) modulates leptin expression in neuronal cells and delineate the signaling cascade. Our study highlights and characterizes a novel salutary role of Vitamin D3 in neuronal cells as it increases the expression of leptin, a pleiotropic cytokine in the brain known to thwart neurodegenerative cascades and augment neuronal survival.

Support or Funding Information

This work was supported by a Grant from the NIH (R01AG045264) to Dr. Othman Ghribi.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
17β‐Hydroxysteroid dehydrogenases and neurosteroid metabolism in the central nervous system
Authors: yang song‐yu , He Xue‐Ying , Dobkin Carl
Published: June 2021

17β‐Hydroxysteroid dehydrogenases are indispensable for neurosteroidogenesis. Neurosteroids are synthesized de novo in neurons and glia from cholesterol transported into mitochondria, or by conversion from proneurosteroids, e. g. dehydroepiandrosterone (DHEA) and pregnenolone, through the same metabolic pathway as de novo neurosteroidogenesis. Proneurosteroids are generated from endocrine glands—the adrenal cortex, ovary, and testis—and then transported into neurons and glia via the blood circulation. Non‐hormonal neurosteroids bind to cell surface receptors, e.g., GABAA or NMDA receptors and elicit antidepressant, anxiolytic, anticonvulsant and anesthetic effects by regulating neuroexcitabilty. Both non‐hormonal and hormonal neurosteroids play important roles in brain development including neuroprotection, neurogenesis and neural plasticity. They are also a critical element in cognitive and memory functions. Mitochondrial 17β‐HSD10, encoded by the HSD17B10 gene mapping to Xp11.2, is found in various brain regions, and is essential for the maintenance of neurosteroid homeostasis. Mutations identified in this gene result in two distinct brain diseases, namely HSD10 deficiency and MRXS10. Their clinical presentations and pathogenetic mechanisms are quite distinct, thus, it is not appropriate to combine them as a single “HSD10 disease”. Since elevated levels of 17β‐HSD10 is found in brains of Alzheimer's disease patients and AD mouse model, and this fascinating protein is responsible for the mitochondrial metabolism of neurosteroids such as 5α‐androstane‐3α,17β‐diol and 17β‐estradiol, analysis of the impact of 17β‐HSD10 and its inhibitor, “β‐amyloid peptide (Aβ),” on the metabolism of neuroactive steroids offers a new approach to AD pathogenesis.

Support or Funding Information

NYS Office for People With Developmental Disabilities

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mast Cell Proteases Activate Glia‐Neurons and Release Interleukin‐33 by Activating MAPKs
Authors: Duraisamy Kempuraj , Selvakumar Govindhasamy Pushpavathi , Thangavel Ramasamy , Ahmed Mohammad Ejaz , Raikwar Sudhanshu P. , Zaheer Smita , Iyer Shankar S. , Zaheer Asgar
Published: June 2021

Parkinson's disease (PD) is characterized by inflammation‐dependent dopaminergic neurodegeneration in the substantia nigra in the brain. Inflammatory mediators released from activated microglia, astrocytes, neurons, T‐cells and mast cells mediate neuroinflammation and neurodegeneration in the brain. 1‐methyl‐4‐phenylpyridinium (MPP+), a toxic metabolite of the parkinsonian neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induces the release of inflammatory mediators from glial cells, neurons and inflammatory cells including mast cells. Mast cells play an important role in neuroinflammation and neurodegeneration. Mast cell proteases are implicated in neuroinflammation. However, the exact mechanism involved in this process is not yet clearly understood. In the present study, mouse bone marrow‐derived mast cells (BMMCs) and mouse fetal brain‐derived glia/neurons and astrocytes were incubated with MPP+, mouse mast cell protease‐6 (MMCP‐6) and MMCP‐7 in vitro. Inflammatory cytokine interleukin‐33 (IL‐33) released from these cells was quantitated by enzyme‐linked immunosorbent assay. MMCP‐6 or MMCP‐7 stimulates astrocyte and glia/neurons to release significantly more IL‐33 as compared with untreated control cells. MPP+ also induced IL‐33 release from mouse mast cells and glia‐neurons. Our results show that mast cell proteases and MPP+ activate mitogen‐activated protein kinases (MAPKs) p38 and extracellular signal–regulated kinases1/2 (ERK1/2) and release IL‐33 from these cells. We suggest that mast cell interaction with glial cells and neurons during neuroinflammation can be explored as a new therapeutic target for PD and other neuroinflammatory conditions.

Support or Funding Information

This work was supported by Veteran Affairs Merit Award I01BX002477 and National Institutes of Health Grants AG048205.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhancing β‐cell Sensitivity to Kisspeptin via Linkage to GLP‐1
Authors: Aragaki Adam K. , Weber Craig , Vagner Josef , Lynch Ronald
Published: June 2021

Glucagon Like Peptide‐1 (GLP‐1) and Kisspeptin (KP) receptors are co‐expressed on pancreatic β‐cells, and activation of both receptors potentiate glucose stimulated insulin secretion (GSIS), although they appear to work via different downstream signaling pathways. Unlike GLP‐1, KP has a low affinity for receptor activation (EC50 20 nM vs >1 uM) making it difficult to use as a drug without significant side effects. We developed a bivalent ligand composed of GLP‐17–36 and KP‐10 binding domains linked together, to evaluate if linking KP to a high affinity ligand would promote its activity at low concentrations by presumably increasing its local membrane concentration after GLP‐1 binding. This feature may also provide some specificity for targeting to β‐cells which express both receptors. The effects of KP‐10, GLP‐1 and the KP‐10/GLP‐1 bivalent analog on insulin secretion from the INS 832/3 cell line were investigated. KP‐10 by itself had no effect on insulin secretion in the absence of glucose, but more than doubled GSIS (basal, 0.23 +/− 0.07; GSIS 0.60 +/− 0.02 ng/min) with an EC50 of ~25 μM. At saturating doses, KP‐10 (100 μm) and GLP‐1 (100 nM) had similar effects on GSIS, and together as monomers had an additive effect (0.79 +/− 0.07 (ng/min) consistent with activation of independent downstream signaling pathways. The dimer also did not activate secretion in the absence of glucose, but doubled GSIS much like the individual monomers (0.64 +/− 0.01 ng/min); that is, an additive response was not obvious. This finding suggests that either the KP‐10 moiety is not binding or is not active in the bivalent ligand, or that GLP‐1 activity is diminished in the bivalent construct such that the combined effect of GLP‐1/KP‐10 is diminished when compared to the additive effect or the monomers. We have evidence from previous studies that binding of the GLP‐1 moiety within a bivalent construct is decreased relative to monomeric GLP‐11, suggesting an additive response may be present. Currently, we are determining the ability of GLP‐1/KP‐10 to initiate downstream signaling from both ligands to resolve the question: is KP active in the GLP‐1/KP at concentrations where monomeric KP‐10 is not.

Support or Funding Information

Funded by the American Diabetes Association and Juvenile Diabetes Research Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Murine Knock‐in Model for Progranulin‐deficient Frontotemporal Dementia with Nonsense‐mediated mRNA Decay
Authors: Nguyen Andrew D. , Nguyen Thi A. , Zhang Jiasheng , Devireddy Swathi , Zhou Ping , Karydas Anna M. , Xu Xialian , Miller Bruce L. , Rigo Frank , Ferguson Shawn M. , Huang Eric J. , Walther Tobias C. , Farese Robert V.
Published: June 2021

Frontotemporal dementia (FTD) is the most common neurodegenerative disorder in individuals under age 60 and has no treatment or cure. Since many cases of FTD result from GRN nonsense mutations, an animal model for this type of mutation is highly desirable for understanding pathogenesis and testing therapies. Here, we generated and characterized GrnR493X knock‐in mice, which model the most common human GRN mutation, a premature stop codon at arginine 493 (R493X). Homozygous GrnR493X mice have markedly reduced Grn mRNA levels, lack detectable progranulin protein, and phenocopy Grn knockout mice, with CNS microgliosis, reduced synaptic density, lipofuscinosis, hyper‐inflammatory macrophages, excessive grooming behavior, and reduced survival. Inhibition of nonsense‐mediated mRNA decay (NMD) by genetic, pharmacological, or antisense oligonucleotide‐based approaches showed that NMD contributes to the reduced mRNA levels in GrnR493X mice and cell lines and in fibroblasts from patients containing the GRNR493X mutation. The expressed truncated R493X mutant protein was functional in several assays in progranulin‐deficient cells, suggesting that NMD inhibition could be an effective as a therapeutic approach for treating progranulin deficiency caused by the R493X mutation. The GrnR493X mice and associated cell lines provide novel model systems to identify therapies for treating FTD resulting from GRN nonsense mutations.

Support or Funding Information

This work is supported by National Institutes of Health Grants AG023501 (to R.V.F.) and AG047339 (to A.D.N.), VA Merit Award BX002978 (to E.J.H.), the Consortium for Frontotemporal Dementia Research (to R.V.F., T.C.W., E.J.H., S.M.F., and B.L.M.), and the Gladstone Institutes. T.C.W. is an investigator of the Howard Hughes Medical Institute. The Gladstone Institutes received support from a National Center for Research Resources grant RR18928.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovery, Efficacy Testing, and Potential Target Identification of Antibiofilm Compounds
Authors: Goode David , Jackson Caroline , Aber Kayla , Shah Ashna , Hensel Linda
Published: June 2021

Ideal biofilm inhibitors would not affect the growth of bacteria, in general, but would be specific for inhibiting reception of the signals to initiate biofilm formation. In this manner, the inhibitor would not affect the growth of normal flora that are imperative for the health of the host. Compounds that resemble biofilm signaling molecules have the potential to competitively inhibit the receptors from receiving signals. Over 200 novel compounds were synthesized and tested by undergraduate students in a sophomore integrated biology and organic chemistry classroom setting. Compounds that demonstrated a statistically significant inhibition of biofilm formation and that had no bacteriostatic or bactericidal activity in one or more of three species of bacteria (Bacillus subtilus, Staphylococcus aureus, and Pseudomonas aeruginosa) in the hands of the undergraduates underwent further testing. Biofilm inhibition and enhancement was confirmed with a modified crystal violet assay, and growth curve analysis confirmed previous disk diffusion and use‐dilution assay results. Hit compounds against Pseudomonas aeruginosa were further tested in a genetics undergraduate classroom using an engineered quorum sensing modulation assay. These results were confirmed in the research laboratory, which along with computational modeling point to LasR as the possible protein target of the novel compounds in Pseudomonas aeruginosa.

Support or Funding Information

This research is supported by the Mercer University Quality Enhancement Plan: Research that Reaches Out.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Isolation and Characterization of Antimicrobial Compound Isolated from Lichen Parmelia vagans.
Authors: Bondarenko Vladimir
Published: June 2021

This study was aimed to isolate and characterize the antibacterial compounds from lichen Parmelia vagans.

Many pharmaceutical products today have derivatives from living organisms. Recently, because of the deficiency of new antibiotics, we once again turned to the nature as a source of new antibacterial compounds. Lichens have been found to have secondary metabolites which have been found to have various physiological effects including antimicrobial activity. A lichen is a composite organism composed of a photobiont such as an alga or cyanobacterium and a mycobiont, a fungus. In this study, the antimicrobial activity of extracts from a lichen identified as Parmelia vagans was evaluated.

Extracts of Parmelia vagans were initially obtained by using various solvents to find the most efficient extraction of antimicrobial compound(s). We separated a crude ethyl acetate lichen extract using reverse phase chromatography and analyzed the fraction using the Resazurin cell viability assay. We used Staphylococcus aureus in the assay because as it has been shown previously, it has relatively high sensitivity to the antibacterial activity of the lichen extract. We identified two major peaks of antimicrobial activity, one peak contained usnic acid, a well‐characterized antibacterial compound, and the second peak contained a novel compound that usually co‐migrates with usnic acid on TLC plate. It was observed that newly isolated compound had no inhibitory effect on the growth of Escherichia coli, and Klebsiella pneumonia at the tested concentrations, but inhibition of growth was observed in Staphylococcus aureus, Bacillus subtilis, and Streptococcus pneumoniae.

Using reverse phase chromatography we isolated a novel antimicrobial compound that will be further characterized structurally and functionally. It will be tested against various gram‐positive and gram‐negative bacterial strains to identify its bacterial target(s).

Support or Funding Information

The above research project was supported by Touro University Nevada intramural research grant.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Unconventional Secretion of Adipocyte Fatty Acid Binding Protein (FABP4) by Adipocytes
Authors: Josephrajan Ajeetha , Hertzel Ann V. , Mashek Douglas , Kim Do‐Hyung , Imai Shin‐Ichiro , Bernlohr David A.
Published: June 2021

Fatty acid binding protein 4 (FABP4) is a cytoplasmic lipid carrier functioning in adipocytes to facilitate lipolysis. Genetic or pharmacologic depletion of the protein provides protection from insulin resistance, asthma, atherosclerosis and inflammation. Metabolic dysfunction has been considered due to the intracellular functions of the protein. However, recent literature has shown that serum FABP4 levels are elevated in patients with metabolic syndrome. FABP4 is a leaderless protein and the mechanism of its release from adipocytes is unknown. Although FABP4 secretion increased with increasing intracellular free fatty acid (FFA) levels and correlated positively with FFA efflux, the molar amount of FABP4 secreted is less than 1% of total molar FFA efflux implying that the protein does not mediate release of bulk lipid in response to lipolytic stimulation. To evaluate the mechanism of FABP4 secretion, we have used primary mouse adipocytes and the 3T3‐L1 adipocyte cell culture model. Results from inhibitor/activator studies showed requirement for some, but not all components of autophagy. For example, classical autophagic inhibitors such as 3‐methyl adenine or the Class III PI 3‐Kinase VPS34 specific inhibitor attenuated FABP4 secretion, while autophagic inducer N‐acetyl sphingosine induced FABP4 secretion. Moreover, FABP4 secretion was potentiated when lipophagy and lysosomal exocytosis were blocked using chemical inhibitors of lysosomal acid lipase or Bafilomycin A1, respectively. Consistent with these results, genetic knockdown of ATG13 or FIP200 (involved in early events of phagophore formation) attenuated secretion, whereas silencing of Atg5 had little effect on FABP4 secretion. Similar to the adipocyte secretion of eNAMPT, blocking SIRT1 by the chemical inhibitor EX527 attenuated secretion, while activating SIRT1 by resveratrol potentiated secretion. Our results show that FABP4 is secreted through autophagy mediated alternative secretion and regulated in part by SIRT1.

Support or Funding Information

Supported by NIH DK053189 to DAB.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differentiation of spermatogonial stem cell‐derived embryonic stem (SSC‐ES) cells towards retinal ganglion cells
Authors: Lee Tin‐Lap , Suen Hoi Ching Ashley , Qian Yan , Liao Jinyue , Luk Chun Shui Alfred , Chan Wai Yee , Ng Tsz Kin Michael , Leung Kai Shun Christopher
Published: June 2021

Embryonic stem cells (ESCs) have a wide range of clinical applications. However, its ethical concerns cannot be neglected. In this study, we collected spermatogonial stem cells (SSCs) from mouse testes and they were subsequently transdifferentiated to ESC‐like cells through a non‐viral and non‐invasive approach. These SSC‐derived ESC‐like cells exhibited stem cell properties similar to ESCs in terms of morphology and pluripotency gene expression profile. Next, we aimed to generate retinal ganglion cells using spermatogonial stem cell‐derived embryonic stem (SSC‐ES) cells. Following previously established protocols with several modifications, SSC‐ES cells were differentiated towards retinal cell lineage and developed optic vesicles in 7–10 days in in vitro culture, while some cells generated neurites in 13–16 days. These neuron‐like cells were positive for RGC‐specific marker Brn3b and other neural markers, thus confirming their RGC identity. We will compare the regenerative effect in vivo using transplantation assay. These cell populations could provide a novel cell‐based therapeutic option for retinal degenerative diseases.

Support or Funding Information

Health and Medical Research Fund, Food and Health Bureau. Hong Kong SAR Government.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Conformational and Thermodynamic Features of Meibum in Adolescents and Adults with Graft‐versus‐host Disease
Authors: Sledge Samiyyah M. , Blackburn Ryan , Ramasubramanian Aparna , Borchman Douglas , Yappert Marta C.
Published: June 2021

Purpose

Graft‐versus‐host disease (GvHD) is a common complication of allogenic hematological stem cell transplantations that is often associated with dry eye disease (DED) classified as a mixture of meibomian gland dysfunction and aqueous tear deficiency. The temperature‐dependence of conformational changes in human meibum (hM) was measured in a comparative study to elucidate the potential impact of GvHD on hM composition, structure and function with DED.

Methods

Donors received full ophthalmic eye exams and DED was clinically diagnosed. A total of 28 donors without DED were separated into two pools: adolescent (age: 17 ± 1 and 63% male) and adult (age: 42 ± 4 and 73% male). Adolescent (age: 15 ± 1 and 80% male) and adult (age: 45 ± 5 and 30% male) pools from a total of 15 donors affected by both DED and GvHD were also tested. The extracted hM was placed in d‐chloroform and analyzed by Fourier Transform Infrared Spectroscopy (FTIR).

Results

Among the measured parameters, the lipid order (% trans) in hM at 36°C increased significantly from adolescents without DED (35.3 ± 4%) to those affected by GvHD (54.7 ± 4%). A similar trend (33.3 ± 0.09% without DED to 38.0 ± 4% with DED/GvHD) was observed in hM from adults. The change in enthalpy for the order‐to‐disorder transition decreased significantly with DED/GvHD in both adolescents (from 163 ± 10 Kcal/mol without DED to 134 ± 9 Kcal/mol with DED/GvHD) and adults (141 ± 5 Kcal/mol without DED to 126 ± 11 Kcal/mol with DED/GvHD). The phase transition temperature (C°) was significantly higher in adolescents with DED/GvHD (38 ± 1°C) compared to donors without DED (30.4 ± 0.6°C), but the increase was less pronounced in adults with DED/GvHD (31 ± 1°C) relative to those without DED (30.0 ± 0.4°C).

Conclusion

Both age and DED lead to conformational and thermodynamic differences in hM. Increases in lipid order and transition temperatures were observed in hM with GvHD. However, these relative increases were more pronounced in hM from adolescents than from adults. The decrease of change in enthalpy for adolescents was more pronounced compared with adults. The conformational and thermodynamic differences observed with age and, importantly, with DED/GvHD are indicative of compositional changes in hM that could impact tear film stability.

Support or Funding Information

Major support was obtained from the National Institute of Health (NIH) EYO RO126180 (DB); NIH Research Supplements to Promote Diversity in Health‐Related Research EYO RO126180S‐1 (SS); and an unrestricted grant from Research to Prevent Blindness, Inc. New York, NY, USA.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anatomical Findings of fetuses Vertically‐infected with Zika Virus
Authors: Figueredo Luisa Fernanda , Franco‐Zuluaga Jorge Andres , Carrère‐Rivera Camille , Rodriguez‐Silva Daniela Sofia , Akle Veronica
Published: June 2021

Zika virus (ZIKV) is a new TORCHS virus with a recent outbreak in Latin America and south of the United States. The virus can be vertically‐transmitted, with many implications for the placenta and the fetuses. Even though there are multiple cases of malformations in babies born from mothers infected with ZIKV, there is little detail about the effect of the infection in the tissues during development.

In this study, four fetuses of the second and third trimester and a newborn, who died within few weeks, from mothers diagnosed with ZIKV were collected from The Legal Medicine Institute of Bogota in Colombia. The cases were subjected to a complete autopsy and the necropsy results were analyzed. For histologic examination, series of micro‐sections were stained with H&E or processed for immunostaining.

Macroscopic findings included compromise of the placenta characterized by opacity and thickness of membranes. The fetuses presented severe edema in extremities and pulmonary hypoplasia. In addition, one case showed Potter facies and low implantation of ear and nose. The newborn also presented petechiae on the thoracic region. Microscopic examination showed severe endothelial lesions in ovular membranes associated with inflammation and dystrophic calcifications of maternal and fetal sides of the placenta. Additionally, there were signs of severe funisitis in the umbilical cord, and congestion of the lungs and accumulation of plasmocytes in the fetuses.

Preliminary results suggest an association between damage of the placental barrier and facilitation of fetal infection. Also, there is evidence of inflammation throughout the fetus' and newborn's tissues, in all cases. A through examination of all organs would give a better overview of the morphological changes caused by the viral infection, which could help understand the infection and give insights into the mechanisms of transmission, prevention and future treatment strategies.

Support or Funding Information

School of Medicine. Universidad de los Andes. Bogotá ‐ Colombia

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
EFFECT OF STAPHYLOCOCCAL ENTEROTOXINS TYPE A (SEA) AND B (SEB) ON THE NEUTROPHIL DYSFUNCTION DURING STAPHYLOCOCCUS AUREUSINDUCED SEPSIS
Authors: Duarte Ana Paula Ferreira , Pinheiro‐Torres Anelize Sgorlon , de Souza Ivani Aparecida , Antunes Edson
Published: June 2021

Sepsis is a systemic inflammatory disease which affects about 17 million people per year worldwide, with a mortality rate above 50%. Sepsis by gram‐positive bacteria has a higher mortality rate when compared with sepsis by gram‐negative bacteria. However, experimental models that adequately mimic the signs of sepsis by gram‐positive bacteria are limited. An inefficient neutrophils (NE) mobilization in Staphylococcus aureus‐induced sepsis is the main cause of death in patients infected with this bacterium. Staphylococcus aureus pathological effects are strong related to the secretion of staphylococcal enterotoxins (SEs). Recently, we have shown that human eosinophils and mice bone marrow granulocytes incubated with SEs exhibit reduced in vitro chemotaxis and adhesion activity. The present study aims to identify the effects of SEA and SEB on the functional properties of human NE. Blood was collected from healthy volunteers after approval from the local ethics committee (Protocol No 61370616.3.0000.5412). Blood samples were placed on isotonic Percoll solution. After centrifugation, the mononuclear cell layer was removed and the pellet containing erythrocytes and granulocytes was aspirated and subjected to isotonic lysis. After lysis cells (98% NE) were resuspended to 4 × 106 cells/ml and submitted to in vitro incubation with SEA or SEB (100 ng/ml; 2 h). Adhesion assays were carried out in 96‐well plates pre‐coated with recombinant human VCAM‐1 and ICAM‐1 for 30 min in the presence of interleukin‐8 (IL‐8). The NE adhesion was calculated by measuring myeloperoxidase (MPO) activity on adherent cells. NE migration assay was performed using a 48‐well microchemotaxis Boyden chamber. Both SEA and SEB reduces human neutrophil adhesion in VCAM‐1 (Control: 4.7 ± 0.5; untreated NE + IL‐8: 6.7 ± 0.6; SEA treated NE+IL‐8: 4.9 ± 0.8; SEB treated NE + IL‐8: 3.7 ± 0.6*# optic density/NE × 106 cells; ) or ICAM‐1(Control: 4.3 ± 0.5; untreated NE + IL‐8: 6.1 ± 0.6; SEA treated NE+IL‐8: 5.5 ± 1.0; SEB treated NE + IL‐8: 3.7 ± 0.9 optic density/NE × 106 cells) coated plates. Similar results were observed for NE chemotactic activity (Control: 18.7 ± 0.8; untreated NE +IL‐8: 52.9 ± 2.7; SEA treated NE+IL‐8: 28.5 ± 1.4; SEB treated NE + IL‐8: 34.2 ± 1.4 NE per high‐power field). The inhibitory effect of SEA and SEB on human NE in vitro adhesion and chemotaxis suggests a role of these toxins on the neutrophil dysfunction associated with the severe sepsis by Staphylococcus aureus.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
INVOLVEMENT OF TLR4 AND PPAR‐α RECEPTORS IN HOST RESPONSE AND NLRP3 INFLAMMOSOME ACTIVATION, AGAINST PULMONARY INFECTION WITH PSEUDOMOSAS AERUGINOSA
Authors: GUGLIANDOLO ENRICO , Fusco Roberta , Ginestra Giovanna , D'amico Ramona , Bisignano Carlo , Mandalari Giuseppina , Cuzzocrea Salvatore , Di Paola Rosanna
Published: June 2021

Background

Colonization with Pseudomonas aeruginosa (PA), the most common pathogen isolated mainly in patients with cystic fibrosis, is particularly difficult to eradicate and is associated with acceleration of decline in lung function and with poorer prognosis. Host resistance to PA is dependent on the activation of an innate immune response. The initiation of this response requires the recognition of bacterial ligands by signaling receptors. Recently, Toll‐like receptors (TLRs) have been found to have prominent roles in the activation of innate immune responses to infection. PA LPS is recognized by TLR4 and has been shown to induce lung inflammation in vivo. In addition, regulation of this process is essential for proper pathogen clearance and to prevent excessive inflammatory response resulting in tissue damage. This occurs through the production of different cytokines, and the activation of different pathways such as inflammasome activation, which is responsible for the production of IL‐1b. Moreover the airway epithelial cells respond with increased production of proinflammatory cytokines as a result of increased NF‐κB activation. One potential regulator of NF‐κB activation are the peroxisome proliferator‐activated receptors (PPARs), and PPARa is a member of the ligand‐activated nuclear receptor superfamily. Thus, the purpose of the present study was to evaluate the role of TLR4 and PPARa in the pulmonary innate immunity response to PA and in the consequent inflammatory response.

Methods

To evaluate the involvement of TLR4 and PPARa in a PA infection, we used TLR4 KO and PPARa KO mice who received an intratracheal (i.t.) administration of 50 ml of PA strain (106 CFU), thus evaluating if these mice were profoundly susceptible to PA compared to WT mice.

Results

The results of the present study showed that administration of PA worsened the pathophysiology of PA lung disease in TLR4 and PPARa KO mice compared to WT.

Conclusions

The present study demonstrated that TLR4 and PPARa receptors would mediate the earliest control of bacterial replication as well as proinflammatory responses to PA infections, and in particular that PPARa receptors are needed to prevent an excessive inflammatory response, as in the control of the inflammosome complex NLP3 activation.

Support or Funding Information

no funding

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeting Androgen Receptor and TRAIL: A Novel Treatment Paradigm for Breast Cancer
Authors: Xie Yan , Chen Linjie , Abel Peter W. , Wei Taotao , Luo Xu , Tu Yaping
Published: June 2021

Objective

TRAIL (TNF‐related apoptosis‐inducing ligand is a promising cancer therapeutic agent due to its minimal toxicity to normal tissues and remarkable apoptotic activity in tumors. However, most breast cancer cells are resistant to TRAIL‐induced apoptosis. Our objectives are to investigate the underlying molecular mechanisms and to develop strategies to overcome such resistance.

Methods

To identify modulators of TRAIL‐induced apoptosis, we carried out a genomewide siRNA screen. To validate the screening result, we either silenced or overexpressed the identified genes in various breast cancer cells and changes in growth and TRAIL‐induced cell apoptosis were determined in vitro and in an orthotopic xenograft mouse model. Finally, we investigated whether small molecules targeting the identified genes improve the effectiveness of TRAIL‐therapy.

Results

We unexpectedly identified androgen receptor (AR) to be responsible for TRAIL resistance. While AR is classically viewed as the key factor in prostate cancer progression, we found that AR expression levels were markedly elevated in human invasive breast cancer specimens including triple‐negative breast cancers (TNBC) that are highly aggressive with poor prognosis. Importantly, breast cancer cell lines express different levels of AR that correlated with their TRAIL resistance. AR overexpression in MDA‐MB‐231 and MDA‐MB‐436 cells suppressed the TRAIL sensitivity whereas knockdown of AR rendered MCF‐7 and MDA‐MB‐453 cells sensitive to TRAIL‐induced apoptosis. AR overexpression also induced TRAIL resistance in breast tumors in vivo. Further, we observed an upregulation of the TRAIL receptor, Death Receptor 5 (DR5) in breast cancer cells, following the removal or inhibition of AR by its antagonists Casodex and MDV3100. Treatment with AR antagonists also enhanced TRAIL‐ induced breast cancer cell apoptosis.

Conclusion

AR signaling suppresses TRAIL‐induced breast cancer cell apoptosis, in part, by suppressing DR5 expression, and a combination of AR antagonists together with TRAIL may be a novel and effective therapy for TNBC.

Support or Funding Information

The project supported by the National Institutes of Health (Grants R21CA193271 and R01HL116849); the National Natural Science Foundation of China (Grants 31100595 and 31300683)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Response of MET inhibition by Glesatinib (MGCD265) and Sitravatinib (MGCD516) in Non‐small Cell Lung Cancer and Malignant Mesothelioma
Authors: Mirzapoiazova Tamara , Tan Carol , Wang Jiale , Mambetsariev Isa , Mambetsariev Bolot , Kulkarni Prakash , Pozhitkov Alex , Wang Yingyu , Christensen James , Engstrom Lars , Salgia Ravi
Published: June 2021

Aberrant activation of the receptor tyrosine kinase (RTK) MET is frequently involved in malignant transformation and inhibiting its activity in dependent cancers is associated with improved clinical outcomes. Glesatinib (MGCD265) and sitravatinib (MGCD516) are next generation MET RTK inhibitors (RTKIs) and while glesatinib also targets the AXL RTK, sitravatinib inhibits a closely related spectrum of RTKs. The efficacy of these compounds in MET‐dependent non‐small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM) cell lines that differed with respect to MET gene expression was evaluated. Both drugs inhibited cell growth and migratory activity with similar potency in NSCLC H1993 cells with clustered MET amplification and A459 cells with ‘wild type’ MET. Also, MPM cell lines H2373 and H2461 with elevated MET expression showed comparable levels of sensitivity to either drug. Kinomics analysis of the affected pathways suggested that both drugs acted similarly, yet distinct signaling was affected when different cell lines were tested. In particular, cellular pathways associated with transformation were inhibited whereas those promoting cell death were activated upon drug treatment. Furthermore, phosphorylation of RON, which interacts with MET, and the downstream signaling molecules AKT and p44/42MAPK were also inhibited in response to hepatocyte growth factor (HGF) stimulation. Importantly, in a pre‐clinical in vivo xenotransplant mouse model, both drugs caused significant tumor growth inhibition. Our data suggest that discerning the expression and activation status of MET and its associated signaling pathways may help guide treatment decisions concerning targeted therapies for NSCLC and MPM.

Support or Funding Information

The National Cancer Institute of the National Institutes of Health under award number P30CA033572 and Mirati Therapeutics.

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