bluuebunny/crossref_metadata_2025 huggingface dataset:
O‐GlcNAc Transferase Regulates Phenotypic Transformation of Vascular Smooth Muscle Cells in Response to Hyperglycemia
Authors: Raman Priya , Mathias Amy , Patel Aakaash , Singh Daman , Storm Kyle
Published: June 2021

Risks of vascular complications are enhanced two‐to‐four fold in diabetes, accounting for increased morbidity and mortality in these patients. Hyperglycemia is an important risk factor for macrovascular complications associated with diabetes. Diabetic patients exhibit an increased incidence of vascular smooth muscle cell (VSMC) migration and proliferation, a hallmark of VSMC phenotypic switching from the quiescent contractile state to a synthetic proliferative phenotype. Hyperglycemia increases glucose flux through the hexosamine biosynthetic pathway triggering an enhanced signaling via O‐linked N‐acetylglucosamine (O‐GlcNAc) transferase (OGT), the key enzyme catalyzing addition of O‐GlcNAc moieties to proteins. Protein O‐GlcNAcylation is a unique post‐translational protein modification implicated in diabetes and related cardiovascular complications. Previous studies have suggested both cardio‐protective and cardio‐detrimental effects of protein O‐GlcNAcylation in diabetes. However, the precise role of OGT in the etiology of diabetic atherogenesis remains incompletely understood. The goal of the current study was to investigate whether OGT modulates VSMC transition to a de‐differentiated phenotype under hyperglycemic conditions. Immunoblotting experiments revealed that in primary cultures of human aortic smooth muscle cells (HASMC) in vitro, benzyl 2‐deoxy α‐D‐galactopyranoside (BG), a pharmacological inhibitor of OGT, decreased high glucose‐induced OGT and O‐GlcNAc protein expression, and this was accompanied with attenuated PCNA (proliferation marker), reduced vimentin (SM synthetic marker) and increased SM‐MHC (SM contractile marker) expression. Consistent with these findings, deletion of OGT using siRNA gene silencing significantly lowered protein O‐GlcNAc and PCNA expression in glucose‐stimulated HASMC compared with glucose‐treated cells transfected with control siRNA. Notably, under glucose‐stimulated conditions, OGT knockdown enhanced calponin (SM contractile marker) expression in HASMC transfected with OGT siRNA vs cells transfected with control siRNA. Finally, electrophoretic mobility shift assay (EMSA) revealed that specific OGT inhibitors (BG and ST045849) attenuated high glucose‐induced DNA‐binding activity of YY1 and Elk1 (transcriptional repressors of SM contractile phenotype) in HASMC compared to cells treated with glucose alone. Taken together, these data clearly demonstrate that loss of OGT inhibits VSMC phenotypic de‐differentiation in response to hyperglycemia, suggesting OGT as a potential therapeutic target in diabetic atherosclerosis.

Support or Funding Information

American Heart Association ‐ Grant‐in‐Aid 16GRNT31200034

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Butanol extract of Morinda Lucida (BEML) protects against Isoproterenol‐induced myocardial infarction in Wistar rats
Authors: Adenipekun Kehinde , Omóbòwálé Temidayo , Oyagbemi Ademola , Adedapo Adeolu , Ashafa Anofi , Yakubu Momoh , Adejumobi Olumuyiwa , Ola‐Davies Olufunke , Ajibade Temitayo
Published: June 2021

With the rising rates of cardiovascular diseases, increase cost of modern therapeutic drugs, as well as the adverse effects of the modern medicine on the users; there is a surge in the need to search for herbs with excellent antioxidant properties for use in preventive medicine. Morinda Lucida, commonly found in the Tropics, precisely in the rain forest of West Africa; it is an important medicinal plant known for its antioxidant and therapeutic uses. The aim of this study was to investigate the cardioprotective effects of the Butanol extract of Morinda Lucida (BEML) against Isoproterenol‐induced myocardial infarction in Wistar rats. Male albino Wistar rats (n=60) were pre‐treated with BEML for a period of 10 days. Group A (control) received food and water ad‐libitum. Group B was challenged with 70 mg/kg Isoproterenol (ISO) alone. Group C: Isoproterenol + 100 mg/kg BEML. Group D: Isoproterenol + 200 mg/kg BEML Group E: 100 mg/kg BEML alone and Group F: 200 mg/kg BEML alone. Groups B, C and D were challenged with ISO on the 11th day. This study found out that ISO induced severe myocardial injuries associated with significant increase in markers of oxidative stress, as confirmed by elevated Malondialdehyde (MDA), as well as decreased reduced Glutathione (GSH), Glutathione peroxidase (GPx), Glutathione, Glutathione‐S‐transferase (GST), Catalase (CAT), Superoxide dismutase (SOD) relative to ISO alone. Pre‐treatment with BEML prior to administration of ISO significantly reduced serum Creatinine kinase myocardial band (CK‐MB) and Lactate dehydrogenase (LDH and also suppressed the expressions of Cardiac troponin I (CTnI), and nuclear factor kappa beta (NF‐κB), respectively. Pre‐treatment with BEML reduced infarct size and restored cardiac ultrastructural integrity. Gas Chromatography Mass Spectrometry (GCMS) analysis revealed the presence of Phytol, 11‐Eicosenoic acid, Palmitoleic acid, Nonacosylic acid, Methyl Palmitoleate, Stearic acid, Vitamin E (alpha‐Tocopherol), Tocopheryl acetate, 17α‐Hydroxyprogesterone, β‐Amyrin acetate, Alpha‐Amyrin acetate, lupenone, and Sclareol in BEML. Together, we conclude that BEML act as potential protective agent against myocardial infarction and other cardiac conditions through its antioxidant, anti‐inflammatory and free radical scavenging activity. The observed cardio‐protective effect might be due to the presence of arrays of phytonutrients.

Support or Funding Information

None

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardiovascular Responses to Selected Cranial Osteopathic Manipulations in Hypertensive Patients
Authors: Ioudina Marina , Toder Eric , Muyot Aurelio , Salloum Liza , Boo Sue , Concepcion Jhaunden
Published: June 2021

Background and Purpose

Osteopathic cranial manipulations (CM) have been used as a part of therapeutic management of hypertensive patients. However, our knowledge about physiological responses to CM in cardiac patients is limited. A quantitative relationship between applied CM and cardiovascular parameters has not been well established. The purpose of this study was to measure changes in heart rate, autonomic input to the heart by using the heart rate variability test, cardiac contractility by using impedance cardiography, and systemic blood pressure in response to selected CM, namely occipital‐atlantal decompression, occipital‐mastoid decompression, and compression of the fourth ventricle.

Method

Hypertensive 65–75 year old subjects, three men and two women, were involved in this study. Hypertension was controlled by an angiotensin‐converting enzyme inhibitor (3 subjects), calcium channels blocker (1 subject) and beta‐blocker (1 subject). Each subject participated in the experimental (CM) and control (sham) groups. A non‐invasive impedance cardiography method was used to measure changes in cardiac parameters before and after manipulations. Data were recorded and analyzed using BIOPAC Systems, Inc. equipment and software. Blood pressure was measured before and after manipulations by using a sphygmomanometer. All manipulations were performed by the same osteopathic physician.

Results

Cranial and sham manipulations resulted in different responses by the subjects. CM produced a decrease in systolic (5%) and mean arterial (6%) pressures, and revealed a trend towards an increase in sympathetic and decrease in parasympathetic cardiac activity based on heart rate variability test. Sham manipulations did not affect blood pressure and were associated with a trend towards a decrease in sympathetic and increase in parasympathetic cardiac activity. No changes in cardiac contractility were recorded for CM and sham manipulations.

Discussion and conclusion

It appears that cranial manipulations may enhance the antihypertensive effect of medication through the effect on autonomic nervous system. The physiological effect of CM on autonomic control of systemic hemodynamics needs further investigation.

Support or Funding Information

This project was funded by the TUN Research Grant Award

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differences in cell proliferation in the cpk and Pkd1 mouse models of polycystic kidney disease
Authors: Rumschlag Matthew , Holthaus Conner , Ellis Monica , Swayze Emma , Zebolsky Aaron , Larson Erik , Heuvel Gregory Vanden
Published: June 2021

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common potentially fatal single gene disorders, resulting from mutations in either the Pkd1 or Pkd2 gene. Renal pathologies found in ADPKD include cysts resulting from increased fluid secretion, cell proliferation, and apoptosis, with an altered differentiation of the epithelial cells lining the cysts. In humans, ADPKD is thought to result from an inherited mutation in the PKD1 gene, followed by a somatic “second hit” mutation in the normal allele. The mechanism of this second hit is not understood. We analyzed the genomic sequence of human PKD1 and found extensive tandem repeats of guanine capable of supporting guanine quadruplex (G4) structures. G4 DNA is four‐stranded, and it is known to functionally contribute to recombination and mutagenesis. By comparison, mice do not carry extensive G4 repeats in the Pkd1 gene, thus targeted deletion of both Pkd1 alleles are required to generate cysts. However, homozygous Pkd1 null mice are embryonically lethal. Therefore, we have generated mice carrying a kidney specific deletion of the Pkd1 gene, called Pkd1CD. These mice develop rapid cystic disease and die by 14 days of age. Another commonly studied mouse model of PKD is a mutation in the cystin gene (cpk mice), which is a mouse model of ARPKD. These mice have a complete loss of the cystin gene, however, they survive until about three weeks of age. To begin to determine the differences in disease progression between the Pkd1CD mice and the cpk mice, we evaluated cell proliferation in these two models of PKD. While kidneys isolated from Pkd1CD mice showed high levels of cell proliferation in the cyst lining cells, similarly cystic kidneys from cpk mice showed very little cell proliferation in the cyst lining cells. Moreover, we observed differences between PCNA and Ki67 labeling as markers for cell proliferation in both mouse models. Because these mouse models differ significantly from human ADPKD, future studies will include replacing portions of the mouse PKD1 gene with the human PKD1 gene that includes the G4 repeats.

Support or Funding Information

NIH DK100972

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bifidobacterium grown on human milk oligosaccharides produce tryptophan metabolite Indole‐3‐lactic acid that significantly decreases inflammation in intestinal cells in vitro
Authors: Ehrlich Amy M , Henrick Bethany , Pacheco Alline , Taft Diana , Xu Gege , Huda Nazmul , Lozada‐Contreras Michelle , Goodson Michael , Slupsky Carolyn , Mills David , Raybould Helen
Published: June 2021

Bifidobacterium longum subsp. infantis (B. infantis) is a commensal bacterium that colonizes the infant gastrointestinal tract. This bacterium can efficiently utilize the abundant supply of oligosaccharides found in human milk (HMO) to help establish residence and generate bioactive metabolites which may produce beneficial effects by acting directly on the host. We hypothesized that metabolites from Bifidobacterium grown on human milk oligosaccharides would reduce inflammation in the intestine. To test this we first needed to identify metabolite candidates by growing B. infantis on either lactose or HMO and analyzing the metabolites produced using NMR spectroscopy. These metabolites were then compared to those found in Bifidobacterium‐dominated infant fecal samples. Once specific metabolites were identified, we treated intestinal and macrophage cells grown in culture to test if they exhibited an anti‐inflammatory response during endotoxin challenge. Lastly, we sought to find which pathway might be activated. We found that B. infantis grown on HMO produced significantly greater quantities of the tryptophan metabolite indole‐3‐lactic acid (ILA). Significantly higher amounts of ILA were also found in fecal samples from a population of infants with high compared to low abundance of Bifidobacterium species. The direct effects of ILA were assessed in macrophage and intestinal epithelial cell lines. ILA (at concentrations from 1 to 10mM) significantly attenuated lipopolysaccharide (LPS)‐induced activation of NF‐kB in macrophages. Similarly, ILA significantly attenuated LPS‐induced increase in the pro‐inflammatory cytokine IL‐8. ILA acts through the aryl hydrogen receptor (AhR) in intestinal epithelial cells as shown through increased nuclear localization of the AhR as well as increased protein expression of the AhR targeted gene CYP1A1. Activation of the AhR has been shown to exert a multitude of beneficial effects on the host including promoting normal intestinal immune function. These findings suggest that ILA, a predominant metabolite from B. infantis, can protect the epithelium via activation of AhR

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Preventive effects of medium‐chain triglycerides supplementation on the dysfunction of mitochondrial oxidative phosphorylation in skeletal muscle under cachectic condition
Authors: Hirabayashi Takumi , Tanaka Minoru , Matsumoto Tomohiro , Ikeji Takuya , Ono Kohei , Takuwa Miho , Maeshige Noriaki , Kondo Hiroyo , Fujino Hidemi
Published: June 2021

Cachexia is a complex metabolic syndrome associated with underlying chronic diseases and is characterized dysfunction of mitochondrial oxidative phosphorylation in skeletal muscle. The preventive effects of medium‐chain triglycerides (MCT) supplementation on the dysfunction of mitochondrial oxidative phosphorylation in skeletal muscle under cachectic condition have been investigated in the present study. ICR mice were randomly divided into 4 groups; control, lipopolysaccharide (LPS), LPS plus long‐chain triglycerides (LCT), LPS plus MCT supplementation. LCT and MCT oil (The Nisshin OilliO Group, Ltd, Tokyo, Japan) were administered to the LPS+LCT and LPS+MCT groups orally by a catheter for 1 week. Cachexia was induced in the LPS, LPS+LCT and LPS+MCT groups via LPS injection (7.5 mg/kg body weight, i.p.) after the supplementation. LPS resulted in a decrease in succinate dehydrogenase (SDH) activity, which is an indicator of mitochondrial function, and PGC‐1α, which is an indicator of mitochondrial biogenesis, in tibialis anterior muscles. LPS also induced a reduction of total ketone bodies concentration level in blood plasma. Meanwhile, MCT supplementation suppressed the decrease of SDH activity and total ketone bodies concentration. In addition, MCT supplementation increased the level of citrate synthase activity in the muscles. These results suggested that MCT supplementation could prevent dysfunction of mitochondrial oxidative phosphorylation in skeletal muscle under cachectic condition.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Transducers of Physical Activity Consortium (MoTrPAC): Creating a Comprehensive Map of Molecular Changes in Response to Physical Activity
Authors: Williams John P. , Joseph Lyndon ,   the MoTrPAC Consortium
Published: June 2021

The NIH Common Fund initiated the Molecular Transducers of Physical Activity Consortium (MoTrPAC) in December 2016 by issuing 19 grants to 37 Principal Investigators from 23 institutions.

Aim 1

MoTrPAC will assemble a comprehensive map of the molecular changes that occur in response to exercise and provide insights into how they are altered by age, sex, body composition, and fitness level.

Aim 2

To fulfill its overall objective of facilitating investigator‐initiated studies and catalyzing the field of physical activity research, MoTrPAC will develop a user‐friendly database that any researcher can access to develop hypotheses regarding the mechanisms whereby physical activity improves or preserves health.

We provide an update on the progress and overview of the study design to be employed by MoTrPAC to develop a molecular map of signals that are involved in transmitting the tissue‐specific and systemic effects of physical activity in animals and humans.

Support or Funding Information

NIH Common Fund

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Profiling the Post‐Injury Hippocampal MicroRNA Response to Transient Forebrain Ischemia: Sub‐regional Differences Between Cornu Ammonis‐1 and Dentate Gyrus
Authors: Arvola Oiva Jouko , Kaidonis Georgia , Xu Lijun , Griffiths Brian , Stary Creed
Published: June 2021

Objectives

Even brief periods of ischemia can result in delayed neuronal cell death in hippocampal cornu ammonis‐1 (CA1), resulting in impaired memory and learning, whereas neurons in the adjacent dentate gyrus (DG) are relatively ischemia resistant. Understanding the pathophysiological and molecular mechanisms underpinning the selective ischemic vulnerability of the CA1 may translate to novel clinical interventions improving outcome in survivors of cerebral ischemia. MicroRNAs (miRNAs) are short (~18–25 nucleotides long) non‐coding RNAs that modulate translation of target messenger RNAs. To date, the miRNA expression profiles of hippocampal CA1 and DG sub‐regions in response to forebrain ischemia have not been examined. The objective of the present study was to assess and compare post‐injury miRNA expression patterns between CA1 and DG in a rat model of forebrain ischemia.

Methods

All experiments were conducted in conformance with the FASEB Statement of Principles for the use of Animals in Research and Education. CA1 and DG sub‐regions were dissected from rat hippocampi following a 10‐minute forebrain ischemia at 3 time points (3h, 24h, and 48h) and at baseline. Total RNA was extracted from samples using Quick‐RNA™ MiniPrep kits (Zymo Research), and the expression of 423 miRNAs were analyzed using nCounter® miRNA expression panel (NanoString). Successful sub‐regional dissection was validated using RT‐qPCR detection of the DG‐rich gene desmoplakin.

Results

Pronounced differences between CA1 and DG in miRNA expression was observed for several miRNAs, greatest with mir‐329, mir‐219‐2‐3p and mir‐181a, known regulators of ischemic injury. Relative levels of CA1 and DG miRNA expression were greatest in established brain‐enriched miRNAs, including mir‐124.

Conclusions

MiRNA expression profiles in response to transient forebrain ischemia were assessed and compared between hippocampal sub‐regions for the first time. Observed differences in microRNA expression patterns between CA1 and DG may explain selective CA1 ischemic vulnerability. Targeted modulation of select miRNAs identified here may serve as a therapeutic intervention to improve cognition in survivors of cerebral ischemia.

Support or Funding Information

Supported by Finnish Cultural Foundation award to Dr. Arvola and American Heart Association award 14FTF19970029 to Dr. Stary.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Have Some Tea, Feed Your Brain: How EGCG Acts as a Protective Agent in a Parkinsonian Model
Authors: Blackburn Josephine Cetha , Magness Emily , Roberts James L
Published: June 2021

Ferrous iron is important in biological function but can be oxidized into ferric iron, at which point it causes oxidative stress. Ferric iron accumulation leading to oxidative stress is one of the factors that contributes to forms of dementia including that associated with Parkinson's Disease. A compound found in green tea called Epigallocatechin Gallate (EGCG) had been shown to act as a metal chelating factor and can naturally bypass the blood brain barrier. Using EGCG as a potential protective agent against Parkinson's disease development would provide a solution to issues regarding drug administration to the brain. We hypothesize that introducing EGCG to the media will help to prevent the oxidation of naturally occurring iron and prevent oxidative stress, leading to less cell death. In this study we used N27 dopaminergic neurons treated with 6‐ Hydroxydopamine (6OHDA) as a model for Parkinson's disease development. In the 6OHDA model for Parkinson's disease, DMT1, one of the proteins responsible for iron uptake, is upregulated increasing iron influx and stimulating oxidative stress. Prior to exposing the cell cultures to 6OHDA, varying concentrations of EGCG were introduced to determine protectiveness. We determined that the IC50 of 6OHDA (normally 35μM) was increased to 45μM upon protection with 5μM of EGCG, 50μM upon protection with 10μM of EGCG, and 55μM upon protection with 20μM of EGCG. This indicates that EGCG is protective in this Parkinsonian model and could possibly have health benefits in the diet. We are currently conducting experiments to determine how EGCG is preventing oxidative stress in this model.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of a Cell Culture System to Examine the Acclimation, Contraction, and Cytoskeletal Remodeling of A7r5 Smooth Muscle Cells to Microgravity
Authors: Whitenack Kaylee M , Arnold Callie , Gibson Danielle , Fultz Michael
Published: June 2021

Few studies have examined the acclimation and contraction of smooth muscle cells in microgravity. In addition, the cytoskeleton in smooth muscle has not been thoroughly examined habituated to microgravity. Although previous studies have noted that the phenotype of smooth muscle may be gravity dependent, those that have been performed have utilized simulated microgravity. The cytoskeleton has been implicated by several investigators as allowing single cells in vitro to respond to changes in gravity and may serve as a gravity sensor (reviewed in Vorselen et al., 2016) at the individual cell level. This investigation will focus on the effect of microgravity on the alpha‐actin, beta‐actin, and myosin components of the cytoskeleton in resting and contracting A7r5 smooth muscle cells. We have provided evidence of differential remodeling of the alpha‐actin, beta‐actin, and myosin cytoskeletal domains in smooth muscle (Brown et al., 2006, Li et al., 2001, Fultz et al., 2000, Fultz and Wright, 2003, and Thatcher et al., 2011). At rest these are organized into filamentous stress cables that run the length of the cell. Upon stimulation, α‐actin and myosin interact to produce force that results in shorting of the cell. This shortening applies stress on the beta‐actin filaments which then undergo subsequent remodeling into configuration that holds the cell in the shortened state. Alpha‐actin and myosin then undergo disassembly/reassembly into a configuration that is optimized for further contraction and aggregate into podosomes that appear as column‐like structures in the contracted cell. While we have proposed that this remodeling is essential mechanism in the development and maintenance of force in smooth muscle (Li et al., 2001). Others have proposed that pododome formation may be relevant in the invasiveness of smooth muscle that is observed in cardiovascular disease as podosomes formation correlates with cell motility and localized degradation of the extracellular matrix (Burgstaller and Gimona, 2005; Lener et al., 2006; Murphy and Courtneidge, 2011). In collaboration with SpaceTango (Lexington, KY) and the Craft Academy for Excellence in Science and Mathematics at Morehead State University, a cell culture system has been developed that will allow for the culture, visualization, stimulation, and subsequent fixation of A7r5 cells aboard the International Space Station. Upon return to Earth, components of the cytoskeleton will be examined by fluorescent microscopy to investigate if microgravity alters the characteristic remodeling observed on Earth.

Support or Funding Information

This material is based upon work supported by NASA Kentucky under NASA award No: NNX15AR69H.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Electrical field stimulation‐induced vasorelaxation responses decreased in anterior and middle cerebral arteries of endotoxemic rats
Authors: SOYDAN GURAY
Published: June 2021

Introduction

Endotoxic shock is characterized by vascular dysfunction, hypotension and multiple organ dysfunction. Loss of vascular tone is a key factor responsible for death in endotoxic shock. Perivascular innervation has an important role in the regulation of vascular tone. Neural control of the arteries is mediated by adrenergic and nitrergic neurons. The nitrergic component is dominant in brain arteries while adrenergic component is dominant in mesenteric arteries. Lipopolysaccharide (LPS) alters the electrical field stimulation (EFS) responses in mesenteric arteries in vitro. Our aim is to study the alterations of EFS responses of cerebral and mesenteric arteries in endotoxemia in rats.

Material and Methods

LPS (10 mg/kg) were given intraperitoneally to rats to make an animal model os sepsis. Rats were anesthetized with gaseous carbon dioxide and decapitated 20 hours after injection, Basilar, anterior cerebral, middle cerebral and 3rd branch of mesenteric arteries were isolated and mounted in a wire myograph covered with platinum electrodes. After U46619 precontraction, frequency‐dependent EFS responses were obtained using a constant current stimulator.

Results

High K+ responses did not change in brain and peripheral arteries. EFS‐dependent vasorelaxation responses decreased in anterior and middle cerebral arteries in endotoxemic rats. EFS‐induced vasorelaxation responses of basilar artery and vasoconstriction responses of mesenteric arteries did not change in endotoxemic rats.

Conclusions

In this study we demonstrate for the first time that EFS‐induced vasorelaxation responses of some cerebral arteries decreased in endotoxemic rats. This EFS‐dependent alterations could be explained by degeneration of perivascular innervations or an imbalance between adrenergic and nitrergic neurons.

Support or Funding Information

This study was funded by Hacettepe University Scientific Research Projects Coordination Unit (TSA‐2017‐12848).

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 Evolution of Heterocomplexation and Multifunctionality in the S100 Protein Complex Calprotectin
Authors: Harman Joseph , Loes Andrea , Shi Ran , Heaphy Maureen , Harms Michael
Published: June 2021

Calprotectin is a multifunctional complex of S100 proteins A8 and A9. It promotes neutrophil migration to sites of inflammation, kills bacteria via oxidative bursting and transition metal sequestration, and exacerbates the inflammatory response by stimulating proinflammatory cell surface receptors. This extensive multifunctionality is the product of preferential heterocomplex formation between A8 and A9, as individual A8 or A9 homodimers are insufficient to perform all of these functions. Our research aims to understand how modern A8 and A9 proteins evolved from a shared homomeric ancestor to form the highly multifunctional calprotectin complex. When did A8/A9 heterocomplexation evolve? How have key biophysical differences between A8 and A9 homo‐ and heterocomplexes changed over time? What are the functional consequences of evolving preferential heterocomplexation? To answer these questions, we are examining complex formation between both extant and ancestrally reconstructed A8 and A9 proteins. We are also characterizing the biophysics of preferential heterocomplexation by measuring oligomeric state and proteolytic susceptibility for all members of the calgranulin clade (S100 proteins A8, A9, A12, and MRP126). Finally, we are mapping changes in biophysical properties and complex formation to changes in functionality. We have found that all therian A8s and A9s form proteolytically resistant heterocomplexes, while the ancestor of all calgranulins forms a proteolytically resistant homohexamer. Antimicrobial activity appears to be conserved across extant A8/A9 complexes, and we are currently evaluating this functionality for ancestral complexes. This work will help unravel the evolution of key biophysical features that contributed to the evolution of multifunctionality in this important innate immunity protein complex.

Support or Funding Information

This work was funded by the National Institutes of Health (NIH‐T32GM007413). Michael J. Harms is a Pew Scholar in the Biomedical Sciences, supported by The Pew Charitable Trusts.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fiber‐type specificity of cancer cachexia‐induced muscle wasting is phosphorylated p70S6K‐dependent
Authors: Tanaka Minoru , Matsumoto Tomohiro , Sugimoto Ken , Fujino Hidemi
Published: June 2021

The purpose of the present study is to investigate the differences between slow‐ and fast‐twitch muscles in cancer cachexia‐induced muscle wasting.

Twelve male Wistar rats were randomly divided into two groups; control and cancer cachexia‐induced group. The cancer cachexia‐induced rats received an intraperitoneal injection of AH‐130 ascites hepatoma cells. The serum, the soleus and plantaris muscles were collected nine days after injection. Then, the expression levels of TNF‐α, p70S6K, FoxO1, FoxO3a, IL‐6, Atrogin‐1, and MuRF‐1 were measured.

The cachexia‐induced rats resulted in a loss in body weight and muscle mass, and an increase in the expression level of TNF‐α in serum. In addition, the mass in fast ‐twitch muscle decreased significantly higher than that in slow‐twitch muscle. The levels of Atrogin‐1 and MuRF‐1 expression, which are the indicators related to protein degradation, increased in the cachexia‐induced group compared with the control group. However, there were no significant differences in Atrogin‐1 and MuRF‐1expression between slow‐ and fast‐twitch muscles. The expression level of phosphorylated p70S6K, which is one of the indicators related to protein synthesis, decreased in the cachexia‐induced group. In addition, the expression level of phosphorylated p70S6K in the cachexia‐induced group was significantly lower in fast‐twitch than in slow‐twitch muscles.

These results indicated that cancer cachexia‐induced both in slow‐ and fast‐twitch muscle wasting might be caused by increased protein degradation factors whereas preferential fast‐twitch muscle wasting depended on the suppression of phosphorylated p70S6K expression, which was different from slow muscle wasting.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of FADS2 Inhibition in Combination with Increased Dietary n‐3 PUFAs on Skeletal Muscle Protein Turnover in Mice Fed a High Fat Diet.
Authors: Brown Katie , Baker Ella , Hawkins William , Sharma Sunita , van der Merwe Marie , Puppa Melissa
Published: June 2021

Introduction

Polyunsaturated fatty acids (PUFAs) are essential dietary components. They are not only used for energy, but also act as signaling molecules. The delta‐6 desaturase (D6D) enzyme, encoded by the FADS2 gene, is one of two rate limiting enzymes that convert the PUFA precursors – α‐linolenic (n‐3) and linoleic acid (n‐6) to their respective metabolites. Alterations in the D6D enzyme activity alters fatty acid profiles and are associated with metabolic and inflammatory diseases including cardiovascular disease and type 2 diabetes. Omega‐3 PUFAs, specifically its constituent fatty acids DHA and EPA, are known for their anti‐inflammatory capability and may be beneficial in the prevention of skeletal muscle wasting through maintenance of protein turnover.

Purpose

The purpose of this study was to determine the role of PUFA metabolites in skeletal muscle protein turnover, in the presence or absence of FADS2 activity.

Methods

Twenty C57BL6 male mice were fed a high‐fat lard (HFL, 45% fat (mostly lard), 35% carbohydrate and 20% protein, n‐6:n‐3 PUFA, 13:1) diet for 6 weeks. Mice were then divided into 2 groups (n=10 per group). One group remained on the HFL diet, while the other group was fed a diet high in n‐3 PUFAs (HFO, 45% fat (mostly Menhaden oil), 35% carbohydrate and 20% protein, n‐6:n‐3 PUFA, 1:3). Each group was further subdivided with half of the animals receiving an orally administered FADS2 inhibitor (100mg/kg/day). After 2 weeks on their respective diets and treatments, animals were sacrificed and gastrocnemius muscle harvested. Protein turnover signaling were analyzed via Western Blot.

Results

Levels of MTOR and AMPK phosphorylation were measured. There was a trend towards increased MTOR phosphorylation with FADS2 inhibition regardless of diet (p=0.07). There was no difference in phosphorylation of AMPK regardless of diet or inhibitor. We analyzed markers of mitochondrial biogenesis which is closely linked with protein synthesis through PGC1‐α and Cytochrome‐C expression, however no significant differences were associated with either marker across all groups.

Conclusion

We conclude that by inhibiting the FADS2 enzyme, there is potential for increased protein synthesis via activation of the MTOR pathway.

Support or Funding Information

University of Memphis School of Health Studies Faculty 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.
The C‐terminus of CsgE Is Involved In Preventing Protein Aggregation
Authors: Aranda Isamar , Binmahfooz Ashwag , Kaur Kuldeep , Rabi Huda , Yeh Lisa , Jayasinghe Sajith
Published: June 2021

Gram‐negative bacteria, such as E.coli and Salmonella, contain proteinaceous, hair‐like, cell surface organelles known as curli. Curli serve to facilitate cell‐cell interactions and are essential for host cell colonization. Curli assembly involves six proteins, CsgA, CsgB, CsgC, CsgE, CsgF, and CsgG. CsgE and CsgF act as chaperones to prevent the premature aggregation of CsgA and CsgB which are the main protein components of Curli. CsgE and CsgF help transport CsgA and CsgB proteins to the cell surface, where CsgA and CsgB begin to assemble to form Curli. We have observed that CsgE is able to inhibit the aggregation of CsgA, as well as human islet amyloid polypeptide (hIAPP), an amyloidogenic polypeptide that is unrelated to curli. We used four different mutants of CsgE (34C, 58C, 99C and 119C) where the wild type residue at the respective position was replaced by a cysteine, and labeled with the fluorophore IASEDANS together with fluorescence quenching to determine the ability of CsgE to interact with the non‐aggregating rat Islet Amyloid Polypeptide (rIAPP). rIAPP differs from hIAPP by only three amino acids, but does not aggregate to form amyloid and was used to reduce the complicating effects of hIAPP aggregation. In the absence of rIAPP IAEDANS labeled at positions 34, 58, 99 and 119 gave Stern‐Volmer quenching constants of 9.6, 9.2, 12.1, and 7.6 respectively, while in the presence of rIAPP these values were 9.7, 8.7, 12.4, and 6.5. The significant reduction in the quenching constant for position 119 in the presence of rIAPP suggests that the fluorophore at this position becomes protected from quenching. The protection could either be due to a conformational change in CsgE driven by binding to rIAPP, or could be due to residue 119 interacting directly with rIAPP, and suggests that the C‐terminal region of CsgE is involved in its ability to prevent protein aggregation.

Support or Funding Information

1. This work is supported by NIH grant 1R15GM123430‐01.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Live section of the brain
Authors: Bobinac Dragica , Celic Tanja , Bobinac Mirna
Published: June 2021

Learning the basic morphology of the brain and topographic relations between certain parts of the brain is fairly complex task in itself and moreover in the Anatomical atlases there is not enough cross‐sectional view of the brain. Therefore, for a successful study of the brain structure through images in the atlas, a great number of images and schemes of coronal, sagittal and horizontal sections are needed. Through a series of such cross‐sections, the student could reconstruct brain structures and study the mutual relationships of neighboring structures.

To enable such a way of learning we have made a section of the brain and we recorded it on a CD along with an explanation of all visible structure.

We have made a sections of five 10% Formalin‐fixed brains.

Two brains were sectioned to show lateral ventricle located within the cerebral hemisphere, third ventricle and fourth ventricle. Then we have made serial 1 cm thick cross‐sections of one brain in coronal plane. Each side of the section was recorded along with an explanation. One brain was cut in median sagittal plane. After that one cerebral hemisphere was cut in horizontal plane in 2 cm thick serial sections while the second cerebral hemisphere was cut in sagittal plane in 2 cm thick serial sections.

Live section of the brain is very well accepted by the students and is very helpful for better understanding of the brain morphology.

Support or Funding Information

Funded by Faculty of Medicine University of Rijeka

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural characterization of human uridine monophosphate synthase (UMPS) and its spatio‐temporal localization in cells
Authors: Kim‐Holzapfel Deborah M , French Jarrod
Published: June 2021

In humans, uridine monophosphate synthase (UMPS) catalyzes the last two reactions in the pyrimidine de novo biosynthetic pathway, with N‐terminal OPRT domain and C‐terminal OMPDC domain. OMPDC has been reported to dimerize in its active state, whereas the oligomerization and structural characterization of OPRT and UMPS are not known. To understand the regulation of proteins in this pathway, we are investigating the structure of UMPS and its spatio‐temporal localization in the cell. Wild type and mutant UMPS were expressed in BL21 E.coli and purified with affinity chromatography and size exclusion chromatography for structural and biochemical studies. Our results indicate that wild type UMPS equilibrates in two different oligomeric states. In cells, UMPS is known to be a cytosolic protein, however, its localization has not been observed when the pyrimidine nucleotide demand increases. We predict that UMPS gather around the mitochondria to increase flux of intermediates under nucleotide depleted conditions. We transiently transfected FLAG‐tagged UMPS into HeLa cells and observed its localization with the mitochondria, which will be further confirmed with super resolution microscopy. UMPS has become an attractive drug target for cancer, autoimmune disease and antiviral therapeutics among others, therefore, structural characterization and cellular localization of UMPS is crucial to determine the regulatory mechanism of the pathway.

Support or Funding Information

SUNY at Stony Brook

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Plasticity Analysis of Drosophila Sleep Circuit After Thermogenetic Sleep Deprivation
Authors: Velazquez Yoheilly , Dubowy Christine , Sehgal Amita
Published: June 2021

Sleep rebound is a homeostatic process characterized by an increase in sleep drive after sleep deprivation. Previous studies have identified neural circuits in Drosophila implicated in sleep regulation, but whether or not there are structural plasticity changes in these circuits after sleep deprivation, and if there is synaptic connectivity between them is unknown. To answer these questions, we used fluorescent markers to identify neural structural changes following thermogenetic sleep deprivation and further characterize the anatomy of two populations of neurons localized in sleep control regions, ExFl2 neurons and c584‐expressing neurons. Immunostaining of ExFl2 neurons produced strong signals in the fan‐shaped body (FSB) and superior medial protocerebrum, while immunostaining of c584‐expressing neurons produced diffused signals throughout the brain. Total fluorescence of ExFl2 neurons in the FSB showed no difference between the sleep‐deprived group and control. In addition, after developing a protocol for sophisticated analysis of 3D structures of interest, results showed no difference between the groups. Altogether, these results suggest that thermogenetic sleep deprivation does not induce plasticity changes in ExFl2 neurons or c584‐expressing neurons. For future directions, using the same approach in other populations of cells that have been related to sleep, and studying the mechanisms underlying any observed structural plasticity will help to better understand sleep control.

Support or Funding Information

National Heart, Lung, and Blood Institute (NHLBI) NIH (Grant #5R25HL084665), Biomedical Graduate Studies (BGS), PRISE Program (Grant #R25GM096955)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Therapeutic Effects and Mechanisms of Baicalein on Rotenone Induced Parkinson's disease in rats
Authors: Lyu Yang , Zhang Xue , Du Lida , Yang Yulin , WANG JINHUA , Du Guanhua
Published: June 2021

Parkinson's disease (PD) is characterized by motor dysfunction, such as bradykinesia, resting tremors, rigidity, and difficulty with voluntary movement etc. The anti‐PD drugs currently used clinically can only relieve symptoms. Up to now, there is still lack of anti‐PD drugs or pathology to alleviate PD. Baicalein is a flavonoid isolated from Chinese medical herb, Huangqin, which has been found in recent years with significant anti‐PD activity. In this study, we investigated the therapeutic effects and mechanisms of baicalein on rotenone‐induced PD rats. We found that baicalein induced rat PD model due to significantly increase the spontaneous activity; prolongation of the rotarod latency and the retention time of the inclined plate, increased the suspension score, and shorted the latency of the rat model in grid experiment. Further research shows the baicalein neuroprotection is closely relate to that baicalein inhibited rotenone induced oxidative stress in the brain, improved the function of mitochondria of rotenone injury, increased α‐synuclein degradation by enhancing rotenone inhibited autophagy and inhibition of NF‐KB and MAPK pathway activation to prevent brain inflammation. In an addition, we also evaluate the neuroprotective effect of baicalein in the rotenone induced SH‐SY5Y and midbrain primary neuronal cell PD model. Our results show that baicalein enhanced mitochondrial biogenesis, improved fusion and fission of rotenone inhibited mitochondrial, alleviated mitochondrial autophagy dynamics process, and increased the number of mitochondria decreased by rotenone through the CREB and GSK‐3β pathway. Baicalein can significantly inhibited the activation of NF‐KB pathway, inhibition of production and release of its downstream inflammatory protein and exert anti nerve inflammation.

Support or Funding Information

CAMS Innovation Fund for Medical Sciences (CIFMS)(2016‐I2M‐3‐007)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Varicella meningitis : An atypical presentation in an immunocompetent individual with seizures managed with acyclovir
Authors: SENTHILKUMAR UMA , Cheta Jordana
Published: June 2021

Viral meningitis, also known as aseptic meningitis, is the most common cause of meningitis and is responsible for between 26,000 and 42,000 hospitalizations yearly in the United States. Bacterial meningitis affects about 4,000 people each year, while viral meningitis affects about 10 people in every 100,000. The most commonly identified pathogens are enterovirus, herpes simplex virus and human immunodeficiency virus. Varicella, rabies and influenza are other viruses that are rarely responsible for viral meningitis. Varicella Zoster is a relatively common disorder in immunocompromised patients characterized by radicular pain and grouped vesicular eruptions. The typical findings in reported cases of VZV meningitis involve skin manifestations and classical signs of meningitis such as neck stiffness. However, only a few cases of meningitis caused by Varicella Zoster Virus (VZV) have been reported. The incidence of VZV meningitis in immunocompetent patients has been reported to be as low as 0.5%. We report an atypical presentation of VZV meningitis in an immunocompetent patient with headache, dizziness, sequalae of seizures without fever and skin manifestations.

Our patient is a 59‐year‐ old African American male who presented with worsening dizziness, confusion, ataxia, lethargy and left‐sided frontal headache. Magnetic resonance imaging revealed mild to moderate burden of white matter abnormality, nonspecific but most likely due to chronic microvascular ischemic disease. No acute stroke. The next day sedation was weaned off and patient remained seizure free on continuous EEG and was extubated 3 days later the CSF came back positive for an elevated Varicella PCR of 42016. Patient was seen by infectious diseases consultant and was advised a course of acyclovir 10mg/kg intravenous every 8 hours for 14 days. All other antibiotics were stopped. Upon discharge, the patient had fully recovered from his symptoms with no neurologic sequalae.

This unique case illustrates an atypical presentation of VZV meningoencephalitis in an immunocompetent patient that lacks many of the obvious features that usually distinguish the disease. Our patient lacked the vesicular rash, as well as other typical features of aseptic meningitis including nuchal rigidity, headache, photophobia, etc. Additionally, the patient's course was complicated by seizure, another relatively uncommon feature of VZV meningoencephalitis. VZV infection or reactivation is, rightfully, most commonly associated with the vesicular rash and prodromal pain that usually accompanies it.

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