bluuebunny/crossref_metadata_2025_split huggingface dataset:
Impact of GPER, Sex, and Age on Arterial Stiffness and Fibrotic Gene Expression
Authors: Kilanowski-Doroh Isabella M. , Ogola Benard O. , Harris Nicholas R. , Gentry Kaylee , Satou Ryousuke , Lindsey Sarah H. 
Published: April 2020

Arterial stiffness is associated with cardiovascular risk independent of blood pressure. Previously, we showed that pulse pressure, a measure of arterial stiffness, is higher in female mice with global deletion of G protein‐coupled estrogen receptor (GPER) despite similar blood pressure. Additionally, we found that the GPER agonist, G1, provides protection from arterial remodeling. Therefore, the current study assessed the impact of GPER, sex, and age on arterial stiffness and aortic expression of four profibrotic genes: decorin (DCN), endothelin 1 (EDN1), type III collagen α1 (COL3A1), and connective tissue growth factor (CTGF). We hypothesized that global GPER knockout (KO) would increase fibrotic gene expression in females more than males, would be exacerbated with age, and would correlate with increased arterial stiffness.MethodsPulse wave velocity (PWV) was obtained in the carotid artery of 20 week‐old and 52 week‐old male and female wildtype and GPER KO mice (n = 3/group) using a Vevo 1100 high resolution ultrasound. RNA was isolated from aortas using a Qiagen RNeasy Mini Kit, and gene expression was quantified using droplet digital polymerase chain reaction (ddPCR). Statistical analysis was performed using three‐way ANOVA and Pearson r Correlation.ResultsIn females, GPER deletion increased expression of all four profibrotic genes in 20 week‐old mice, but this relationship was reversed in 52 week‐old mice. In males, GPER deletion was associated with lower expression of fibrotic genes in both age groups. PWV significantly correlated with increased expression of CTGF (r = 0.66, p = 0.019) and EDN1 (r = 0.61, p = 0.036). In addition, the expression of CTGF, COL3A1, EDN1, and DCN were positively correlated (p < 0.005). Interestingly, expression of both ERα and GPER were increased with aging in female mice (p < 0.05).ConclusionIncreased PWV was accompanied by upregulation of four profibrotic genes in the aorta. Adult female GPER KO mice had increased stiffness and higher expression of fibrotic genes. However, GPER deletion was associated with lower PWV and fibrotic gene expression in middle‐aged mice. Therefore, the protective effect of GPER in the vasculature was reversed with aging, despite the fact that aging upregulated estrogen receptor expression. Future studies will determine the mechanism by which GPER influences the expression of fibrotic genes and arterial stiffness.Support or Funding InformationNIH HL133619
What Neutrons Read in Tea Leaves: A High‐Pressure, Small‐Angle Neutron Scattering Study
Authors: Spruill Racheal 
Published: April 2020

Green tea polyphenols contribute to its taste, aroma, and health benefits such as reduced risk of degenerative diseases and cancer. 85% of polyphenols in green tea are catechins, mostly Epigallocatechin gallate (EGCG), a potential alternative to synthetic food additives. EGCG has been shown to prevent the growth of bacteria responsible for food spoilage while showing antioxidant activity in food systems. Polyphenols are however susceptible to light, high extraction temperatures, and alkaline conditions, which cause their degradation during extraction and food processing. Several approaches have been used to improve polyphenol yields, stability, and production costs, including various solvent free extraction techniques, modification of the structure of EGCG or association with protein‐based carriers. Bovine serum albumin (BSA), found in blood serum and milk, functions as a transporter of endogenous and exogenous compounds in the circulatory system and has been shown to bind and stabilize polyphenols. The nature of the interactions is thought to be mostly hydrophobic. The underlying mechanisms of BSA‐polyphenol interactions, as well as their stability, are central to the development of effective carriers as delivery systems and to the engineering of improved food processing. While high‐pressure (HP) is increasingly used in sterilization and food processing, HP is also a non‐invasive tool for the study of protein‐polyphenol interactions. This project combines neutron scattering and HP techniques to investigate the structure and stability of complexes of BSA with polyphenols, particularly EGCG. Small‐angle neutron scattering (SANS) is a non‐destructive technique that can be used to probe a broad range of interactions under various concentrations, temperature, pH and pressure conditions. The results highlight how neutron scattering can contribute new insights into polyphenol bioavailability in biological and food‐processing environments.Support or Funding InformationSupport for Racheal Spruill was provided by the Center for High Resolution Neutron Scattering, a partnership between the National Institute of Standards and Technology and the National Science Foundation under Agreement No. DMR‐1508249.
The Structurally Unusual Retaining β‐Kdo Glycosyltransferase WbbB Uses a Double‐Displacement Mechanism with an Intermediate Adduct Rearrangement Step
Authors: Kimber Matthew S. , Forrester Taylor J.B. , Mallette Evan , Ovchinnikova Olga G. , Nothof Jeremy T. , Koizumi Akihiko , Lowary Todd L. , Whitfield Chris 
Published: April 2020

WbbB is a modular, trifunctional glycosyltransferase (GT) which synthesizes lipopolysaccharide O‐antigen in Raoultella terrigena. The two C‐terminal WbbB GT domains build the polysaccharide repeat, while the N‐terminal GT domain acts as a terminator, adding a β‐Kdo (3‐deoxy‐d‐manno‐oct‐2‐ulosonic acid) residue to O3 to rhamnose as required for O‐antigen export. This N‐terminal GT domain is highly unusual in sequence and was not recognized as a GT by bioinformatics tools. The structure shows the dual Rossmann‐fold motifs characteristic of GT‐Bs, but with extensive deletions, insertions and rearrangements result in a unique architecture. The CMP‐binding site, however, retains motifs homologous to GT‐B sialyltransferases. WbbB is a retaining GT, transferring Kdo from the CMP‐®‐Kdo donor with net retention of anomeric configuration. All well‐characterized retaining GTs seem to employ a front‐side SNi substitution mechanism. This is somewhat surprising, as analogous retaining glycosyl hydrolases use a double‐displacement mechanism; here an essential catalytic acid residue attacks the donor saccharide to form a covalently bonded intermediate, with a second acidic residue acting as a general base to hydrolyse this adduct. We show, using mass spectrometry, that WbbB forms a Kdo adduct with Asp232 and its variants (D232N or D232C), while a D232A variant is both wholly inactive and forms no adduct. The x‐ray structure of D232N CMP‐®‐Kdo donor complex shows that the anomeric carbon of the donor is inaccessible to any potential acceptor but is instead positioned immediately in contact with, and in line with, the carboxylate of Asp232. Structures of D232N‐ and D232C‐Kdo adducts show that the Kdo adduct is rearranged into a second half‐site, interacting with a distinct set of catalytic residues. A ternary complex of D232C‐Kdo plus a synthetic acceptor shows that Glu158 forms direct hydrogen bonds with O3 of rhamnose, positioned adjacent to and in‐line with the anomeric carbon of Kdo, activating it for attack. Glu158 is also essential, with variants being wholly inactive. Together, this shows that WbbB uses a glycosyl hydrolase‐like double‐displacement mechanism. We propose that crowding around the anomeric carbon by the carboxylate group likely precludes retaining ulosonic acid transferases using the more straightforward SNi mechanism, while adduct rearrangement avoids having the acceptor compete with the leaving nucleotide for access to the anomeric carbon of the donor.Support or Funding InformationThis work was funded by an NSERC Discovery grant (04045‐2015) to MSK and support from the Canadian Glycomics Network (SD‐1) to TLL; TJBF is the recipient of a University of Guelph Graduate Excellence Entrance Scholarship and an Ontario Graduate Scholarship.
The Herpes Simplex Viruses Utilize a Recombination‐Dependent Replication Mechanism to Replicate Viral Genomes
Authors: Weerasooriya Savithri , DiScipio Katherine A. , Peterson Jaliyah , Weller Sandra K. 
Published: April 2020

Although it has been known for decades that HSV replication results in the formation head‐to‐tail concatermers, the mechanism by which concatemers form is not understood. One long‐standing model involves circularization of incoming viral genomes followed by rolling circle replication. However, several lines of evidence suggest that HSV DNA replication may be more complex, relying instead on a recombination‐dependent replication mechanism. The HSV single‐strand DNA binding protein (SSB), ICP8, is the central player in all stages of DNA replication. Additionally, ICP8 can promote efficient annealing of complementary ssDNA and is thus considered to be a single strand annealing protein (SSAP). HSV encodes a conserved alkaline nuclease (UL12) that interacts with ICP8 to form a complex that is reminiscent of evolutionarily conserved phage recombinases, such as lambda red a/ß. We hypothesize that ICP8 and UL12 interact to promote recombination‐dependent DNA replication to generate head‐to‐tail concatemers necessary for the production of infectious virus. Due to the multifunctional nature of ICP8, it has been difficult to tea1se out the role of ICP8 as a SSAP from the various roles it plays during viral replication as a classical SSB. We have recently identified a novel ICP8 mutant, Q706A/F707A, that lacks annealing activity but retains many of the other functions characteristic of classical SSBs. Interestingly, this mutant is unable to synthesize viral DNA, indicating that the annealing function of ICP8 is essential for viral DNA synthesis (Weerasooriya, DiScipio et al. PNAS 2018)Annealing is a complex process requiring many types of molecular interactions. We hypothesize that it involves both ICP8‐DNA and ICP8‐ICP8 interactions as well as conformational changes induced by the recognition of complementary DNA. Based on their proximity to Q706A/F707A, we suggest that annealing may be mediated by a cluster of conserved residues (residues 701 to 719) in the shoulder domain of ICP8. We have identified five conserved arginine residues (R701, R704, R712, R713 and R714) in this region. Alanine mutagenesis of these residues showed that all mutants, except R704A are unable to complement the growth of a null mutant suggesting that these residues are important for viral growth. Biochemical characterization of purified mutant and WT proteins will be presented along with models for how ICP8 promotes efficient annealing during DNA synthesis.Support or Funding InformationNIH funding AI069136 and AI021747 to S.K.W and F30AI143125 to K.A.D
PGRMC1 Stabilizes Cytochromes P450 And Is Required for Activity In Vivo
Authors: McGuire Meredith R. , Mosher Eric , Myers Stephanie , Bumpus Namandje , Espenshade Peter 
Published: April 2020

Progesterone receptor membrane component 1 (PGRMC1) is a membrane‐bound hemoprotein with many ascribed functions that binds cytochromes P450 in yeast and human cells. In this study, we generated a whole‐body Pgrmc1 KO mouse to examine the function of PGRMC1 in cytochrome P450 biology. Here, we report that Pgrmc1 is a novel post‐translational stabilizer of cytochromes P450 required for cytochrome P450 activity in vivo. In mouse liver, Pgrmc1 bound 13 cytochromes P450 of diverse families (Cyp1, 2, 3, 8, and 51) and functioned to maintain liver cytochrome P450 levels post‐transcriptionally. In the absence of Pgrmc1, cytochrome P450 protein levels were 22–70% of wild‐type. These reductions in P450 enzymes were shown to be physiologically important. Pgrmc1 promoted Cyp1a2 activity in vitro as the Vmax for the Cyp1a2‐dependent O‐deethylation of 7‐ethoxycoumarin was 40% lower in the absence of Pgrmc1 in liver membranes. Mechanistically, PGRMC1 stabilized CYP1A2 post‐translationally, doubling the half‐life of the cytochrome P450 in transfected human cells. The stabilization of cytochromes P450 and promotion of cytochrome P450 activity by Pgrmc1 were the result of a protein‐protein interaction between Pgrmc1 and the cytochrome P450 that did not require Pgrmc1 to bind heme. Reductions in Cyp2e1 protein in Pgrmc1 KO mice protected animals against hepatotoxicity during Cyp2e1‐dependent acetaminophen overdose, the leading cause of acute liver failure in the U.S. This study establishes PGRMC1 as a central component controlling cytochrome P450 activity thus making PGRMC1 the first new player in the liver cytochrome P450 system in 50 years as it joins cytochrome P450 oxidoreductase and cytochrome b5. This study also establishes PGRMC1 as a clinically relevant effector of cytochrome P450 activity, which has direct clinical implications related to the efficacy of pharmacotherapies and will be of interest to pharmacologists, clinicians, and the pharmaceutical industry.Support or Funding InformationM.M. was supported by NIH 5T32GM007445, NIH F31HL131185, and American Heart Association 15PRE25250008. P.E. was supported by NIH R01HL077588 and NIH R21HL094774.
Novel Control Mechanism of the Neurovascular Coupling in an Alzheimer’s Disease Model
Authors: Puebla Mariela , Fritz Elsa , Álvarez Alejandra , Figueroa Xavier 
Published: April 2020

Alzheimer’s disease (AD) is a type of dementia related to age characterized by a progressive and irreversible deterioration of memory and general cognitive abilities. There is a consensus that the production of β‐amyloid protein (Aβ) aggregates correlates with neuronal alterations, triggering the characteristic neurodegeneration process of this disease. Furthermore, disease progression has been closely associated with cerebral vascular alterations, such as reduction in cerebral blood flow observed in patients and animal models of AD.It has been documented that Aβ not only has harmful effects at neuronal level but has also been associated with the alteration of astrocytes normal activity. One of the events modulated by astrocytes at cerebral level is its key role in the neurovascular coupling (NVC), mechanism that ensures nutrients and oxygen supply in proportion to the increase in neuronal activity. The NVC functions controlled by the astrocytes is mediated by vasodilator signals, a process that is dependent of intracellular calcium increase in astrocytes, which is propagated and coordinated by the release of paracrine and autocrine adenosine triphosphate (ATP).There is no consensus about the way of ATP release from astrocytes during NVC, but the description of a new channel, Calcium Homeostasis Modulator 1 (CALHM1) which permit the ATP release in some type of neurons, could be an important element in astrocyte signaling during this mechanism. In previous results we observed that ATP is released through CALHM1 in an astrocyte primary culture model stimulated with glutamate (a stimulus validated in NVC studies) and also was dependent of the CALHM1 activation by S‐nitrosylation through nitric oxide (NO) produced by the astrocytes.Considering that it has been reported that NVC is altered in AD, which contribute to the pathophysiological mechanisms of this disease, our main aim was to evaluate the ATP release in astrocytes primary cultures from a validated murine model of AD: B6C3‐Tg(APPswe,PSEN1dE9)85Dbo/Mmjax.Our results show that ATP release, and CALHM1 relative levels and expression are significatively reduced in astrocytes primary cultures from AD animals in comparing with cultures from wild type animals. These results suggest that calcium signaling could be diminished in the astrocytes in AD and could explicate the relation between the alteration of NVC in AD.Support or Funding InformationUniversidad del Desarrollo Intern Proyect 23.400.163
Therapeutic Vulnerabilities in Acute Myeloid Leukemia (AML) with TP53 Alterations
Authors: Ogunsan Oluwateniayo , Antony Marie Lue , Noble-Orcutt Klara E. , He Fiona , Sachs Zohar 
Published: April 2020

Acute myeloid leukemia (AML) is a cancer of the blood and bone marrow with a low two‐year survival rate. Standard chemotherapy achieves complete remission in 60–80% of patients, but 20–30% patients relapse due to leukemia stem cells (LSCs) that can self‐renew and recapitulate disease. AML with TP53 alterations has a lower prognosis with survival rates of 0–10% at one year. In vitro drug screens have identified drugs to target AML, but none have studied the effect of these therapies on LSCs with TP53 alterations. These in vitro drug screens include Crizotinib, Elesclomol, AZD1480, GW2580, Venetoclax, and Entrectinib. Crizotinib, AZD1480, GW2580, Venetoclax, and Entrectinib are signaling pathway inhibitors. Elesclomol induces oxidative stress and apoptosis in cancer cells. The goal of this research is to understand the molecular mechanisms of self‐renewal and therapeutic vulnerabilities in LSCs of AML with TP53 alterations. Specifically, we investigate whether these agents target LSCs from AML with TP53 alterations. We performed in vitro viability and in vitro colony forming assays (CFAs) on primary human AML samples with TP53 alterations, plating them with drugs or vehicle control. Furthermore, we used CYTOF to assess the effects of each drug on signaling within leukemia subpopulations. Crizotinib proved to be effective in reducing in vitro viability, in vitro CFAs, and affecting the expression of immunophenotypic markers, such as CD34 and CD123, which are markers for leukemia stem cell populations. Our data reveals cell‐type specific effects of Crizotinib in human AML with TP53 alterations. Our findings suggest that Crizotinib may be an effective therapy for patients with AML with TP53 alterations.Support or Funding InformationThis research was supported and funded by: NIH/NIGMS MARC U* STAR T34 HHS 00026 National Research Service Award to UMBC, Summer Research at the University of Minnesota Medical School NIH HLBI 2R25HL088728‐11A1, Dr. Colin Campbell, Dr. Craig Henke, American Cancer Society Mentored Research Scholar Grant (MSRG‐16‐195‐01‐DDC), Frederick A. Deluca Foundation, CTSI K to R01 Award, NIH/NCATS, Lois and Richard King Assistant Professorship in Medicine, Division of Hematology, Oncology, Transplantation, Dept. of Medicine, University of Minnesota University of Minnesota Foundation Donors
Identification and characterization of a novel Anoctamin 1 inhibitor and its anticancer effects
Authors: Jo Sungwoo , Seo Yohan , Namkung Wan 
Published: April 2020

Anoctamin 1 (ANO1 also known as TMEM16A) is a calcium‐activated chloride channel that is amplified in various carcinomas including prostate cancer, esophageal cancer, breast cancer, and pancreatic cancer, and its overexpression promotes cell proliferation and cell migration in various cancer cells. A cell base‐screening revealed that (E)‐1‐(7,7‐dimethyl‐7Hfuro[2,3‐f]chromen‐2‐yl)‐3‐(1H‐pyrrol‐2‐yl)prop‐2‐en‐1‐one (Ani‐E1) is a novel, potent and selective ANO1 inhibitor with an IC50 value of 1.23 μM. Ani‐E1 showed 144 times stronger activity on ANO1 inhibition than ANO2 inhibition and did not alter the chloride channel activity of CFTR and the intracellular calcium signaling. Notably, Ani‐E1 strongly decreased cell viability of PC‐3 and FaDu cells expressing high levels of ANO1 with a decrease in ANO1 protein levels. In addition, Ani‐E1 significantly enhanced apoptosis via activation of caspase 3 and cleavage of PARP in PC‐3 and FaDu cells. This study shows that a novel ANO1 inhibitor, Ani‐E1, can be a potential candidate for the treatment of cancers overexpressing ANO1, such as prostate cancer and esophageal cancer.Support or Funding InformationWe thank the National Research Foundation of Korea (NRF‐2017R1A2A2A05069364 and 53 NRF‐2018R1A6A1A03023718, NRF‐2019R1I1A1A01061117) for generous financial support. This research was supported (in part) by Yonsei University Research Fund (Post Doc. Researcher Supporting Program) of 2019 (project no.: 2019‐12‐0013).
Proximal tubule albumin uptake – potential role for endothelin system in sickle cell disease mice
Authors: Kasztan Malgorzata , Alrefai Hasan , Taylor Crystal , Pollock David M. 
Published: April 2020

Elevated plasma endothelin‐1 (ET‐1) reported in sickle cell disease (SCD) patients correlate with microalbuminuria, a major mortality risk factor in SCD. ET‐1 contributes to glomerular and tubular injury in SCD, as evidenced by increased glomerular permeability to albumin and urinary excretion of tubular injury markers. Although selective ETA receptor antagonism reduces albuminuria in humanized sickle cell (HbSS) mice, the mechanism of this action remains unclear. The aim of the study was to determine whether selective ETA receptor antagonism prevents albuminuria by preserving the expression of tubular albumin‐associated transporters in proximal tubule (PT) cells. Male C57BL/6 or HbSS and genetic control (HbAA) mice were used to determine the effect of ET‐1 on the expression of proximal tubule albumin trafficking mediators, such as megalin and NHE‐3. Exposure of primary mouse PT cells to ET‐1 (50nM) for 8h decreased megalin (53% reduction) and doubled NHE‐3 expression. Pre‐treatment with the ETA antagonist, BQ123 (1 μM), preserved megalin expression and had no effect on NHE‐3. Moreover, selective ETB receptor blockade (BQ788, 1 μM) prevented ET‐1‐mediated increase in NHE‐3 expression. Primary PT cells isolated from HbSS mice showed decreased megalin mRNA expression as well as protein abundance relative to HbAA PT controls. Ten‐week treatment with selective ETA receptor antagonist (10mg/kg/day) preserved expression of megalin at control levels. There were no differences in NHE‐3 mRNA expression in HbSS PT cells regardless the treatment. Interestingly, PT cells from HbSS cultured with HbSS plasma and ET‐1 (10nM) had decreased NHE‐3 protein abundance compared to non‐treated cells. These results potentially uncover a novel role for ET‐1 in PT albumin handling and suggest that PT ET‐1 receptor signaling contributes to albuminuria in SCD.Support or Funding InformationSupport provided by an NIH U01 HL117684 to D.M.P, AHA 19CDA34660073 and NIH‐NHLBI K99HL144817 to M.K.