bluuebunny/crossref_metadata_2025 huggingface dataset:
PC4 Coactivator Mediates Sp1‐driven Luteinizing Hormone Receptor Transcription
Authors: Liao Mingjuan , Zhang Ying , Dufau Maria L.
Published: June 2021

The luteinizing hormone receptor (LHR) has an essential role in sexual development and reproductive function by participating in gonadal maturation and regulation of steroidogenesis and gametogenesis. Transcription of the LHR gene is subject to several modes of regulation by multiple effectors through its core promoter, which contains two activating Sp1/Sp3 binding domains and an inhibitory motif recognized by EAR2 and EAR3/COUP‐TF1. The present studies demonstrate that PC4 acts as a coactivator of Sp1 to mediate Sp1‐dependent LHR expression. In MCF‐7 breast cancer cells and choriocarcinoma JAR cells, knockdown of PC4 expression with its specific siRNA significantly reduced LHR promoter activity. Co‐transfection of PC4 with an Sp1 or Sp3 expression plasmid showed that PC4 synergistically enhanced Sp1 but not Sp3‐activated LHR expression in reporter gene analyses, indicative of a functional interaction between PC4 and Sp1. In support of this, DNA affinity precipitation assay revealed the sequence‐specific association of PC4 with Sp1/Sp3 binding sites of the LHR gene promoter. Moreover, dephosphorylation of PC4 by calf intestinal phosphatase substantially increased its interaction with the Sp1 binding sites, suggesting a role of phosphorylation in modulation of the PC4/Sp1 interaction. Taken together, these studies have demonstrated that PC4 functions as a coactivator of Sp1 in LHR regulation.
Connecting mechanism of proteotoxicity: from worm to mouse
Authors: Dillin Andrew
Published: June 2021

The greatest risk factor for nearly all neurodegenerative diseases is aging. The central hypothesis of our work is that continual production of aggregation prone proteins eventually leads to age onset proteotoxicity and disease. The key area of research to address is to understand the molecular mechanisms that prevent proteotoxicity during early life that become compromised with age. To address this question, I will present our results in C. elegans that point towards a protective disaggregation activity and a protective aggregation activity that are regulated by the aging program. Next, I will describe our work in which we have discovered the same protective mechanisms exist in mammals. Finally, I will broaden the scope of my presentation to illustrate that other pathways of aging also regulate age onset proteotoxicity in simple and more complex model systems such as C. elegans and mice and elaborate their mechanism of action.
A systematic study of the structural and regulatory features of post‐translational modifications of cellular enzymes: a case study based upon information in PhosphoSitePlus™
Authors: Kornhauser Jon Mark , Skrzypek Elzbieta , Zhang Bin , Murray Beth , Tkachev Sasha , Hornbeck Peter V
Published: June 2021

PhosphoSitePlus (PSP) is an open resource offering comprehensive information about site‐specific protein modification. PSP aggregates all published sites of protein post‐translational modifications, as well as thousands of newly‐discovered modification sites identified using high‐throughput methods at CST and elsewhere. Data that is manually curated includes upstream events that regulate modifications, and downstream consequences of modifications on the protein and more global cellular processes. The vast amount of information in PSP can be mined to build signaling networks to explore novel features of cellular processes.

Of >10,000 proteins curated into PSP, about 1,850 are cellular enzymes with >15,000 known modification sites. We have analyzed information in PSP for patterns and interactions that may provide novel insights into the biology and biochemistry of enzymatic systems. These studies include:

1) pathway analysis of regulatory information in PSP;

2) potential associations between specific upstream kinases and classes of modified enzymes, protein domains shared between enzymes, or novel sequence motifs;

3) identification of sites within motifs or domains that are common to related enzymes; and

4) role of modification sites common to sets of enzymes on regulation of downstream processes.

www.phosphosite.org

Supported by NIH grants R43GM065768, R44AA014848, and 5R44CA126080
Expression and Purification of Mammalian PCSK9 and Characterization of its Prodomain for Extracellular Trafficking.
Authors: Zhong Xiaotian , Cameron‐Landis Lora , Stochaj Wayne , Awad Tony , Pocas Jennifer , Stahl Mark , Kriz Ron
Published: June 2021

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted member of the proprotein convertase (PC) family that is responsible for the proteolytic maturation of secretory precursors to bioactive proteins and peptides involved in many physiological processes. Recent studies reveal that PCSK9 plays a critical role in cholesterol metabolism by regulating low density lipoprotein receptor (LDLR) protein levels and function, consistent with the links of its naturally occurred mutations to hypercholesterolemia and hypocholesterolemia. In this study, we have expressed rat PCSK9 in HEK293 cells to a level of 3mg/L for a structural and functional analysis. The protein was purified to homogeneity and found active in down‐regulating surface expression of LDLR. Yet the purified protein possesses no protease activity, likely due to the fact that PCSK9 contains a pro‐domain not only essential for its extracellular trafficking but also tightly associated with its catalytic groove. Taking advantage of the recent finding that the PCSK9 prodomain can work in trans, we further characterized this structural element by deleting its C‐terminal residues, inserting protease cleavage motifs to engineer a second processing event, and swapping with prodomains of other PC family members. The results indicate that the prodomain of PCSK9 can tolerate the residue insertions but is sensitive to the C‐terminal deletion, and possesses unique features different from other PC family members.
Oleic acid reduces lipopolysaccharide‐induced expression of iNOS and COX‐2 through inhibition of reactive oxygen species and NF‐¥êB activation in BV2 murine microglial cells
Authors: Oh Young Taek , Kang Insug
Published: June 2021

Microglial cells are the major cell types involved in neuroinflammation, resulting in brain tissue damages during cerebral ischemia and neurodegenerative disorders. In this study, we examined the effects of the monounsaturated fatty acid oleic acid (C18:1, OA) on LPS‐induced proinflammatory mediators and the mechanisms involved in BV2 microglial cells. OA inhibited LPS‐induced the expression of iNOS and COX‐2 mRNA and proteins as well as the production of NO and prostaglandins. We showed that OA inhibited LPS‐induced NF‐κB activation and phosphorylation of inhibitor κB kinase (IKK). We also showed that OA inhibited LPS‐induced phosphorylation of Akt and p38 and PI 3‐kinase activation, but not that of ERK. Finally, we showed that OA reduced the reactive oxygen species (ROS) accumulation and an antioxidant N‐acetylcysteine inhibited NF‐κB activation and IKK phosphorylation in LPS‐stimulated BV2 cells. Taken together, our results suggest that OA has an anti‐inflammatory effect by inhibiting ROS generation and NF‐κB activation in LPS‐stimulated BV2 microglial cells. This work was supported by grants from Korea Science and Engineering Foundation.
Studies on the role of acid sphingomyelinase and ceramide in the LPS‐induced TNFa secretion by macrophages
Authors: Rozenova Krasimira Aleksandrova , Deevska Gergana , Nikolova‐Karakashian Mariana N
Published: June 2021

The objective of this study is to decipher the role of ASMase in LPS‐triggered TNFα production. ASMase deficient (ASMase (‐/‐)) and wild‐type (ASMase (+/+)) mice and macrophages were stimulated with LPS and TNFα levels in serum and medium were monitored by ELISA and western blot analysis. The data revealed that ASMase deficiency is linked to increased production of soluble TNFα in response to LPS but does not affect MAP kinases activity or the rate of TNFα mRNA synthesis. These results suggest that ASMase affects post‐transcriptional processing of TNFα.

26kDa TNFα precursor (proTNFα), is cleaved to the 17kDa soluble form (sTNFα) by the TNFα converting enzyme (TACE). Substantial portion of the un‐cleaved proTNFα appeared to be rapidly degraded in the endosomes/lysosomes as judged by the increase of proTNFα in the presence of lysomotropic agent ammonium chloride. These data suggest that TACE is a rate‐limiting step in TNFα secretion. Notably, TACE activity is 2‐3 fold greater in ASMase(‐/‐) macrophages as compared to control cells, and it is inhibited by the addition of ceramide. Further studies revealed that the level of TACE mRNA was not affected but ASMase deficiency influences the sub‐cellular distribution of TNFα and TACE.

Together, these results suggest that the rate of post‐translational processing of TNFα is important regulator of TNFα secretion and it is modulated by ASMase activity.
Oxidized L‐A‐phosphatidylcholine B‐arachidonoyl‐gamma‐palmitoyl (oxPAPC) induces Increased Synthesis of mRNA and protein expression for IL‐6 in Human Aortic Endothelial Cells
Authors: Dvoracek Lucas A , Kreisberg Jeffrey I , mckinney jordan m , schmid gabrielle , francis Amanda d , francis Amanda d , lee Huey Miin , detrick melinda s , kreisberg robert
Published: June 2021

Oxidized L‐A‐phosphatidylcholine B‐arachidonoyl‐gamma‐palmitoyl (oxPAPC) induces Increased Synthesis of mRNA and protein expression for IL‐6 in Human Aortic Endothelial CellsLucas A. Dvoracek1, Jeffrey I. Kreisberg1, Jordan McKinney1, Gabrielle Schmid1, Amanda D. Francis1, Huey Miin Lee, Ph.D.2, Melinda S. Detrick1, and Robert Kreisberg1

1West Liberty State College, West Liberty, West Virginia 26074

2West Virginia University, Morgantown, WV 26506‐6330

L‐A‐phosphatidylcholine B‐arachidonoyl‐gamma‐palmitoyl (PAPC), a bio‐active component of low density lipoprotein, can become oxidized in the blood. OxPAPC may play a role in triggering an inflammatory reaction at the endothelial cell level. Atherosclerosis, the build‐up of plaque in the vascular wall, may be exacerbated by chronic inflammation initiated by the presence of oxPAPC. In this study, human aortic endothelial cells were treated for 6h with 50μg/mL of PAPC, oxPAPC, or neither. Cellular lysates were collected and treated for mRNA isolation. Transcripts were amplified by RT‐qPCR using primers specific for Interleukin‐6 (IL‐6). Interleukin‐6 has been identified as a potentially sensitive indicator of cardiovascular disease. Data from the RT‐qPCR technique suggests a 23‐fold up‐regulation of IL‐6 mRNA for cells treated with oxPAPC over both PAPC‐treated and No Treatment groups. This publication was made possible by NIH Grant Number 2P20 RR016477 from the IDeA Networks of Biomedical Research Excellence Program of the National Center for Research Resources, WVNASA, and WVEPSCoR SURE
Phosphatidylinositol polyphosphates: ionization constants in fluid phosphatidylcholine model membranes
Authors: Kooijman Edgar Eduard , Gangoda Mahinda , Kumar Satyendra , Gericke Arne
Published: June 2021

Phosphatidylinositol polyphosphates (PIPs) are only a minor pool of membrane phospholipids but are involved in many intracellular signaling processes including membrane trafficking, cytoskeleton remodeling, receptor signal transduction and nuclear events. Phosphoinositide properties are largely determined by the characteristics of their headgroup, which at physiological pH is highly charged but also capable of forming hydrogen bonds. Many proteins have developed special binding domains that facilitate specific interaction, while other proteins interact with phosphoinositides via non‐specific electrostatics. Despite its importance, only limited information is available about the ionization properties of phosphoinositides. We have investigated the pH dependent ionization behavior of all three naturally occurring phosphatidylinositol bisphosphates as well as of phosphatidylinositol‐3,4,5‐trisphosphate in mixed multi‐lamellar phosphoinositide/phosphatidylcholine vesicles using magic angle spinning 31P‐NMR spectroscopy. Aside from the detailed pH dependent titration behavior, the current study also provides compelling evidence for mutual PIP2 interaction without the presence of cationic proteins. The ionization behaviors of these PIPs solve several historical contradictions and will have important implications for the physiological processes in which they are involved.
Structural and Binding Study on the Interaction of Small Molecule Antagonists with the EphA4 Receptor
Authors: Qin Haina , Shi Jiahai , Noberini Roberta , Pasquale Elena B. , Song Jianxing
Published: June 2021

As a promising class of drug targets, the Eph receptor tyrosine kinases regulate a variety of physiological and pathological processes. The EphA4 receptor plays important roles in the inhibition of the regeneration of injured axons, synaptic plasticity, platelet aggregation and cancer. In this study, we determined the first crystal structure of the EphA4 ligand‐binding domain, which adopts the same jellyroll β‐sandwich architecture as EphB2 and EphB4. Isothermal titration calorimetry, NMR spectroscopy and computational docking were further employed to characterize the binding between EphA4 and small molecules, which antagonize ephrin‐induced effects in EphA4‐expressing cells. These molecules bind to the EphA4 ligand‐binding domain with Kd values in a µM range. NMR HSQC titrations show that these molecules significantly perturb EphA4 residues located at D‐E loop, E β‐strand and J‐K loop. Molecular docking shows that by interacting with these residues, small molecules occupy a cavity in the high‐affinity ephrin binding channel of EphA4. Our results demonstrate that the ephrin‐binding channel of the Eph receptors is amenable to targeting with small molecule antagonists and suggest avenues for further optimization.
Adapting to student learning styles: using cell phone technology in undergraduate biology, chemistry, and biochemistry instruction
Authors: Timpte Candace , Sauder Deborah , Pennington Richard , Tsoi Mai Yin , Paredes Julia Barker , Pursell David P.
Published: June 2021

Students of biology and chemistry traditionally make 3x5 flash cards to assist learning nomenclature, structures, and reactions. Advances in educational technology over the past ten years have enabled flash cards viewed on computers, offering an endless array of drilling and feedback for students. The current generation of students is less inclined to use computers, but they use their cell phones 24 hours a day. This report outlines these trends in student educational technology use and reports on our novel use of educational technology, that of using cell phone flash cards to help students learn biology, chemistry, and biochemistry nomenclature, structures, and reactions. Students responded positively to cell phone flash cards in a pilot study and a more detailed study in general chemistry, general biology, organic chemistry, and biochemistry sections is ongoing and results will be reported.
Antibiotics that Kill
Authors: Reguieg Zahia , Jones Tynier , De la Cruz Alba , Frink Monique , Carpenter Aaron , Fisher Thomas , Lynch Alexis , Hope Kelly , Stevens Danielle , Bommakanti Ananth , Zengel Janice M
Published: June 2021

Ribosomes, organelles that carry out protein synthesis, are composed of ribosomal RNA and proteins and are divided into two subunits. The small subunit decodes genetic information while the large subunit helps form the peptide bonds of the newly created proteins. Ribosomes are the target for some antibiotics; if the ribosome cannot function, the bacteria cannot make proteins. Ribosomal mutations can cause antibiotic resistance that decreases the drug's activity. Antibiotics have been overused, and bacteria are becoming resistant to them. Two antibiotics, chloramphemicol and erythromycin, inhibit protein synthesis. Chloramphemicol inhibits peptide bond formation while erythromycin can block the tunnel of the large ribosomal subunit preventing the new proteins from exiting the ribosome. Mutations in ribosomal proteins can cause cells to be resistant to the effects of these drugs. The Patterson SMART (Students Modeling a Research Topic) Team, in collaboration with UMBC and MSOE, designed and built a 3‐D physical model of the ribosome using 3D printing technology to show how the ribosome interacts with antibiotics. Supported by a HHMI Precollege Science Education grant.
Structural analysis of OmpR a transcription regulator in the mutualistic bacterium, Xenorhabdus nematophila
Authors: Borden W. , Wolff B. , Vogt C. , Jordan P. , Ayesh M. , Brook A. , Day J. , Elewa M. , Flesner G. , Kuriga L. , Lopez H. , Maier B. , Radke J. , Yacoob Z. , Klestinski K. , Vogt D. , Forst Steve
Published: June 2021

A partnership between Marquette University High School students participating in the MSOE SMART Team (Students Modeling A Research Topic) program and a researcher enabled the team to explore structure and function, and to build a physical model using 3D printing technology, of OmpR. OmpR is a transcription factor necessary for the nutrition gathering strategy of the bacterium Xenorhabdus nematophila. Bacteria absorb food through membrane pores, which change in size to optimize food intake and to protect themselves from toxins.

X. nematophila has sensor proteins in the outer membrane. When food touches an outer membrane receptor (EnvZ), the receptor transfers phosphate to OmpR forming OmpR‐P, which can bind to DNA. When nutrients are abundant, OmpR‐P binds to OmpC, a gene promoting formation of a small pore, allowing food yet limiting influx of toxins. When food is scarce, OmpR‐P binds OmpF, a gene promoting formation of a large pore, allowing more food intake and growth of a flagellum enabling movement to a nutrient rich location.

The OmpR gene is also responsible for production of antibiotic compounds that combat a broad range of microorganisms. X. nematophila often forms a mutualistic relationship with nematodes. The bacterium‐nematode pair seek to inhabit and eventually kill certain insects, benefiting from the nutrients provided by the insect's corpse.

Supported by a grant from NIH‐NCRR‐SEPA
Somatic Cell Nuclear Transfer to Create a Miniature Swine Model of Retinitis Pigmentosa
Authors: Ross Jason W , Zhao Jianguo , Walters Eric M , Samuel Melissa , Narfstrom Kristina , Jeong Manbok , DeMarco Paul J , McCall Maureen A , Kaplan Henry J , Prather Randall S
Published: June 2021

Retinitis pigmentosa is a group of genetic eye conditions for which the most common autosomal dominant form is a P23H substitution in the rhodopsin gene. Our objective was to create a P23H rhodopsin model in miniature swine with different founding animals demonstrating variation in disease severity. We used a 17.5 kb fragment of the human genome containing the P23H affected coding sequence and genomic regulatory regions. The DNA fragment was transfected into fetal fibroblast of NIH miniature swine with or without a selectable marker. Clonal cell colonies with stable integration were utilized for somatic cell nuclear transfer. Six transgenic founder animals were produced from five pregnancies. Clinical examination at 0.5, 3 and 6 months and a standard ISCEV ERG protocol at dark‐ and light‐adapted levels was used at 3 (n=6) and 6 months (n=2) of age to assess phenotype. No fundus abnormalities were detected although significant variation in ERG response was observed among founders. At 3 months, two founders had severely compromised dark‐ and light‐adapted responses. Changes in the two founders evaluated again at 6 months indicated disease progression. Phenotype variation in the founders is likely due to integration site and copy number and may not be affected by the utilization of a co‐transfected marker gene. Support: NIH U42‐RR18877 and R24EY015636, USDA 35205‐05309, Discovery Eye Foundation.
Developing team‐centered independent research skills in an advanced biochemistry laboratory course
Authors: Savage Thomas J , Roberts Linda M
Published: June 2021

Traditional biochemistry laboratory courses emphasize exercises designed to reinforce concepts presented in lecture courses and to expose students to common techniques in biochemical studies. However, other skills required for biochemistry graduates (particularly those starting careers in industry) include experimental design, ability to work independently and the ability to work in teams to achieve scientific goals. Thus, we modified an advanced biochemistry laboratory course to allow students to design and execute independent experiments in a team setting centered on heterologous expression and purification of his‐tagged proteins. Each semester, the class as a whole has a goal to express and purify protein for structural or kinetic studies. The class is organized into teams, and each team is responsible for developing a protocol for a different variant of the protein of interest, and each individual student project focuses on optimizing a specific secondary purification column. Students work in their teams to explicitly define their team and individual goals and to write progress reports throughout the semester. Attitudinal surveys completed before and after implementing the team‐centered approach indicate students value the team experience while appreciating learning fundamental protein purification skills.
Correlation of Poly(ADP‐ribose) Polymerase (PARP) with Markers of Oxidative Stress in Human Atherosclerotic Plaques: Pathobiological Determination of Atherosclerosis in Youth (PDAY) Study
Authors: Hans Chetan Parkash , Troxclair Dana , Zerfaoui Mourad , Naura Amarjit , Zieske Arthur W. , Malcom Gray T. , Strong Jack P. , Boulares Hamid
Published: June 2021

We have established a strong correlation between PARP activation and markers of oxidative stress in a mouse model of atherosclerosis. The present study presents evidences establishing an active role for PARP in human atherosclerosis. Human aortic atherosclerotic plaques (AHA grade 2 and 3) obtained from the PDAY study were subjected to immunostaining to detect iNOS (increased nitric oxide production), nitrotyrosine (nitration of proteins), 8‐oxo‐dG (oxidative DNA damage), PAR (PARP activation), active caspase‐3 (apoptotic cell death) and CD‐68 (macrophage/foam cells). These markers were selected to test the hypothesis that nitration of protein induced by increased nitric oxide production leads to DNA damage and PARP activation which ultimately participates in cell death and inflammation in atherosclerosis. PARP activation was localized within and in the vicinity of plaques suggesting active role of this enzyme in plaque dynamics. PARP activation correlated strongly with the presence of nitrotyrosine, iNOS and 8‐oxo‐dG as analyzed by double immunofluorescence. Moderate correlation was observed between PAR and active caspase immunoreactivity. Little correlation between PARP activation and CD‐68 was observed in this study. Overall the data suggest that PARP is activated in human atherosclerotic plaques at early stages and strongly correlates with markers of protein/DNA damage and cell death.
IL‐20 is regulated by hypoxia‐inducible factor and upregulated after experimental ischemic stroke
Authors: CHEN WEI YU , CHANG MING SHI
Published: June 2021

IL‐20, an IL‐10 family member, is involved in various inflammatory diseases, such as psoriasis, rheumatoid arthritis, and atherosclerosis. We investigated whether hypoxia in vitro and an in vivo model of ischemic stroke would upregulate IL‐20 expression. In vitro, IL‐20 expression increased in hypoxic HaCaT , HEK293 cells, chondrocytes, monocytes and glioblastoma cells. Inhibiting HIF‐1alpha inhibited CoCl2‐induced IL‐20 expression. We identified two putative hypoxia response elements in human il20‐gene promoter. Promoter activity assays showed that CoCl2‐mimicked hypoxia activated luciferase reporter‐gene expression. In vivo, experimental ischemic stroke upregulated IL‐20 in the sera and brain tissue of rats. IL‐20 stained positively in glia‐like cells in peri‐infarcted lesions, but not in contralateral tissue. Administration of IL‐20 monoclonal antibody ameliorated ischemia‐induced brain infarction of rats after experimental ischemic stroke. In vitro, RT‐PCR analysis showed that glioblastoma cells GBM8901 cells expressed IL‐20 and its receptor subunits IL‐20R1, IL‐20R2, and IL‐22R1. IL‐20 also induced production of IL‐1beta, IL‐8, and MCP‐1 in GBM8901 cells. We conclude that IL‐20 was responsive to hypoxia in vitro and in the ischemic stroke model, and that upregulation of IL‐20 in the ischemic brain may contribute to brain injury.
Appropriately glcosylated beta1‐integrin serves as an E‐selectin ligand
Authors: Lin Regina Junhui , Chong Siew‐Meng , Lim Yaw‐Chyn
Published: June 2021

Introduction

The interactions between selectins and their ligands are important for the extravasation of leukocytes into target tissues.

Aim

To identify adhesion molecules that mediate E‐ and P‐selectin interactions under defined flow conditions.

Methods

The interactions of NB‐DLB‐1 cells (a lymphoma cell line) with E‐ or P‐selectin were examined in parallel plate flow chamber studies. Putative selectin ligands were identified by function blocking assays, affinity capture and Western blot.

Results

NB‐DLB‐1 cells interact with E‐ and P‐selectin in a shear‐dependent manner under defined flow conditions. Function blocking assays identify PSGL‐1 and CD44 as the major P‐selectin ligands; and L‐selectin and beta1‐integrin as the E‐selectin ligands. Affinity capture using E‐selectin/IgG reveals that the E‐selectin‐binding beta1‐integrin is of the high molecular weight glycoform(s).

Conclusion

NB‐DLB‐1 cells interactions with E‐ and P‐selectin are mediated by different adhesion molecules. Our data show that L‐selectin and the extensively‐glycosylated form(s) of beta1‐integrin are E‐selectin ligands on NB‐DLB‐1 cells. This novel function of beta1‐integrin implies that in addition to the classical VLA‐4‐VCAM‐1 interactions, appropriately glycosylated beta1‐integrin can bind to E‐selectin.

Study funded by ARF, NUS and NMRC, Singapore.
THE FREQUENCY OF MICRONUCLEI IN EXFOLIATED BUCCAL CELLS IN INDIVIDUALS WORKING WITH PHOTOCOPYING MACHINES
Authors: ELEZAJ ISA REXHEP , LETAJ KASUM RRUSTEM , SELIMI QERIM ISAK , GASHI UJEZA SALI , JONUZI SUZANA
Published: June 2021

To study the effects of occupational exposure of photocopying machines, exfoliated buccal cells from 22 individuals (males), working with photocopying machines and control subjects ( 20 males), were examinated for micronucleus (MN) frequency. Analysis of buccal MN frequency reveal that in individuals working with photocopying machines were significantly higher (P<0.001), compared with that in control subjects. Our findings indicate that photocopying machine workers are under risk of significant cytogenetic damage.
VEGFR‐2 Targeted Microbubble Contrast Agents for Ultrasound Molecular Imaging of Tumor Angiogenesis
Authors: Anderson Christopher R , Rychak Joshua , Backer Joseph , Ley Klaus , Klibanov Alexander
Published: June 2021

Ultrasound molecular imaging is an emerging technique for detecting intravascular targets. We have developed a novel microbubble (MB) ultrasound contrast agent covalently coupled to a recombinant single‐chain VEGF construct (scVEGF), which binds VEGFR‐2 (KDR) on the angiogenic endothelium. Ligand conjugation was first validated with a fluorophore (BODIPY‐cystine), and covalently bound dye was detected by fluorometry and flow cytometry. MB were subsequently conjugated to scVEGF, which bound at ~100,000 molecules/MB (ELISA). Targeted adhesion of scVEGF‐MB was demonstrated with in vitro flow assays. At 0.5 dynes/cm2, scVEGF‐MB exhibited 5‐fold higher adhesion to both recombinant murine VEGFR‐2 and porcine aortic endothelial cells transfected with VEGFR‐2 (PAE/KDR) compared to non‐targeted control MB. Additionally, scVEGF‐MB targeted to immobilized VEGFR‐2 in an ultrasound flow phantom showed an 8‐fold increase in mean acoustic signal relative to casein‐coated control channels. A murine model of colon adenocarcinoma was used to assess targeted adhesion in vivo, and scVEGF MB, but not control MB, showed significantly higher ultrasound contrast signal enhancement in tumors. These results demonstrate the functionality of a novel scVEGF‐bearing microbubble contrast agent, which could be a valuable research tool for molecular imaging of VEGFR‐2. Supported by NIH 1R43EB007857.
Utility of QDot® bioconjugates in the detection of ERG gene re‐arrangements in prostate cancer
Authors: Nagy Dea , Nagle Raymond , Sathyanarayana Ubaradka , Riley Janice , Murillo Adrian , May Eric , Jiang David , Santa‐Cruz Ashley , Richard Jeanne , Lefever Mark , Kosmeder Jerry , Farrell Michael , Bieniarz Chris , Miller Phil , Pestano Gary
Published: June 2021

Prostate cancer (PCA) is one of the most prevalent malignancies affecting men worldwide. Recent discovery of ERG gene re‐arrangements in PCA and their strong association with disease progression has therapeutic and diagnostic implications. Gene fusions identified thus far are characterized by 5' genomic regulatory elements ( TMPRSS2), fused to the ETS family of transcription factors (TF), that can lead to over‐expression of oncogenic TF. In this report, we have developed an automated assay that includes novel FISH probes in conjunction with Qdot‐antibody conjugates, to detect the various ERG re‐arrangements. We have shown sensitive and specific detection of gene rearrangements in xenografts, H1660, LnCAP, and VCAP. These data confirmed previous reports of homozygous intronic deletion, no deletion, and varied deletions of 5'ERG region of Chr 21 in the respective xenografts. Imaging with FISH filters we observed robust signal at 20x magnification. This study outlines the development of novel gene probes and fluorescent bioconjugates that enable simultaneous detection of the 5' and 3' ERG translocations in PCA.
The Epstein ‐ Barr virus Latent Membrane Protein 1 and Transforming Growth Factor beta1 synergistically induce Epithelial‐to‐Mesenchymal Transition in human alveolar epithelial cells
Authors: Sides Mark D , Klingsburg Ross , Gordon Kristan , Shan Bin , Flemington Eric , Lasky Joseph P
Published: June 2021

Idiopathic Pulmonary Fibrosis (IPF) is characterized by thickening of the interstitium due to collagen deposition resulting in destruction of the alveolar architecture. A growing body of evidence suggests lung myofibroblasts are derived from epithelial origin through Epithelial‐Mesenchymal Transition (EMT). TGF‐b induces EMT in lung epithelial cells in a time and dose dependent manner. Higher occurrence of Epstein‐Barr virus (EBV) has been reported in lung tissue of IPF patients compared with that of non‐IPF patients. Our data show a remarkable increase in mesenchymal cell markers with concurrent reduction in the expression of epithelial cell markers in lung epithelial cells co‐treated with LMP‐1 and very low doses of TGF ƒÒ. We show that the pro‐EMT LMP1 signaling is primarily through the NF‐kB pathway and the pro‐EMT TGFb signaling is through the ERK pathway. LMP1 reduces TGFb signaling through Smad2/3 and increases TGFb ERK signaling. Together, these data suggest that the presence of EBV‐LMP1 in lung epithelial cells sensitizes these cells to TGF ƒÒ, synergistically inducing EMT. Our in vitro data may help explain the observation that IPF patients demonstrating positive staining for LMP‐1 in alveolar epithelial cells have a more rapid demise than patients in whom LMP‐1 is not detected.
Lung Pathology Following Instillation of Metal Nanoflake Particulates
Authors: Kristovich Robert Lee , Horsmon Michael , Moran Theodore , Taylor James Tracy
Published: June 2021

Sprague‐Dawley rats were exposed through intratracheal instillation, in accordance with an IACUC approved animal use protocol (ECBC protocol #07‐395), to a low, medium or high dose of copper, aluminum or brass nanoflake, synthesized through a ball‐milling procedure. At various time points post exposure the animals lungs were either recovered for histopathological analysis or broncheoalveolar lavaged for pro‐inflammatory cytokine analysis or differntial cell count analysis. Our results have indicated a different mechanism of toxicological action based on the underlying chemistry of the instilled particulate. Specifically, the copper and brass nanoflakes are soluble under ambient lung conditions. This has led to the observation of an acutely inflammatory lung response following instillation of even low levels (0.1 mg/kg) of these nanoflakes within 24 hours post exposure; that is resolved by 14 days post exposure. The aluminum nanoflake is insoluble and therefore must undergo macrophage clearance from the lung following instillation. Interestingly, the kinetics of inflammatory reaction of the lung are much slower with the aluminum nanoflake and the observed response is increasing out to at least 14 days post exposure. This seems to indicate a more chronic problem associated with aluminum nanoflake exposure when compared to the acutely toxic response observed with copper and brass nanoflakes.
Long Term Improvement in mdx Cardiomyopathy after Therapy with Peptide‐conjugated Morpholino Oligomers
Authors: Willis Monte S , Jearawiriyapaisarn Natee , Kole Ryszard
Published: June 2021

The cardiomyopathy found in Duchenne Muscular Dystrophy (DMD) is responsible for death due to heart failure in ~30% of patients. Strategies to bypass DMD‐causing mutations to allow an increase in body‐wide dystrophin have proven promising, but increasing cardiac dystrophin continues to be challenging. The purpose of this study was to determine if therapeutic restoration of cardiac dystrophin improved the significant cardiac hypertrophy and diastolic dysfunction identified in mdx dystrophin‐null mice. mdx mice were treated with an arginine‐rich cell‐penetrating peptide‐conjugated phosphorodiamidate morpholino oligomer (PPMO) that delivered a splice‐switching oligonucleotide‐mediated exon skipping therapy to restore dystrophin in mdx mice. PPMO successfully restored cardiac dystrophin expression, preserved cardiac sarcolemma integrity, and significantly reversed the cardiac pathology found in mdx‐null mice. By echocardiography and Doppler analysis, we identified that PPMO treatment ameliorated the cardiac hypertrophy and diastolic dysfunction identified in mdx mice and provided a durable cardiac improvement for up to 6 months. These results demonstrate for the first time that PPMO‐mediated exon skipping therapy early in the course of DMD may effectively retard the mdx associated cardiac hypertrophy and diastolic dysfunction with significant long‐term impact.
PI3‐kinase activation attenuates the TLR2‐mediated macrophage proinflammatory cytokine response to F. tularensis infection
Authors: Medina Edward A. , Morris Ian R. , Berton Michael T.
Published: June 2021

We sought to clarify the role of the PI3‐kinase/Akt pathway in regulating early (< 9 h) proinflammatory responses in macrophages infected with Francisella tularensis Live Vaccine Strain (LVS). LVS‐induced TNF‐α and IL‐6 secretion was markedly increased in macrophages from wildtype C57BL/6 mice pre‐exposed to the PI3‐kinase inhibitor wortmannin compared with vehicle pre‐exposed macrophages; the levels of phosphorylated p38 MAPK and ERK1/2 were also enhanced and remained elevated for a longer period of time. LVS infection rapidly induced MAPK phosphatase‐1 (MKP‐1) expression in a TLR2‐ and PI3‐kinase‐dependent manner. Peak levels of MKP‐1 correlated closely with the decline in p38 MAPK and ERK1/2 phosphorylation. MAPK activation and cytokine production were entirely TLR2‐dependent. Surprisingly, PI3‐kinase/Akt activation was TLR2‐independent; it was also TLR4‐, TLR9‐, TIRAP/Mal‐ and TRIF‐independent. PI3‐kinase activation was, however, dependent on MyD88. These data suggest that LVS infection restrains the TLR2‐triggered pro‐inflammatory response of macrophages via parallel activation of the PI3‐kinase pathway, which enhances MKP‐1 expression. This results in the accelerated deactivation of MAPKs and suppression of proinflammatory cytokine production. This inhibitory pathway may be activated by a novel MyD88‐dependent pattern recognition receptor that is engaged by LVS. This work was supported by NIH grant AI057986.
TNFa Antagonizes PDGF‐AB Chemotactic Effects on Mesenchymal Stem Cells (MSC) Migration
Authors: Phua Andy Yong Han , Vijayakumar Praveen , Choolani Mahesh , Chan Jerry , Lim Yaw‐Chyn
Published: June 2021

MSC are a promising cell source for cellular therapy but often, systemic delivery to target tissues is sub‐optimal. Aim: This study examines the effects of pro‐ and anti‐inflammatory mediators and growth factors on MSC migration in vitro.

Methods

The effects of various chemokines/cytokines, growth factors and lipid mediators (used alone or in combinations) on MSC migration was determined using transwell assays. After 5hr of incubation, the inserts were fixed and stained with Giemsa stain. 21 random fields were digitized at 100x magnification and the mean number of transmigrated cells/field determined.

Result

Compared to media control (14±5 cells), MSC migration was significantly increased in the presence of PDGF‐AB (59±21 cells) and IGF‐1 (28±13 cells) whereas SDF‐1, bFGF, SLC, GM‐CSF, VEGF, LTB4 and LXA4 had no detectable chemotactic effect. We found that TNFa significantly inhibited MSC migration (7±2 cells). Furthermore, the addition of TNFa together with PDGF‐AB into the bottom well also significantly inhibited the PDGF‐AB induced migration by 41.3±11.3% (p<0.05 as compared to PDGF‐AB alone).

Conclusion

TNFa significantly inhibits both baseline and PDGF‐AB induced MSC migration in‐vitro. Our data suggest that the precise composition of the cytokine/chemokine milieu at the injury site will greatly influence MSC recruitment into the tissue.

This research is funded by URC NUS.
a‐synuclein interaction with the dopamine transporter stimulates a transporter mediated Cl‐ current
Authors: Chirwa Sanika , Goodwin Shawn J , Swant Jarod , Khoshbouei Habibeh
Published: June 2021

The neuronal plasma membrane dopamine (DA) transporter (DAT) is essential for the maintenance of DA homeostasis in the brain. Both DAT and a‐synuclein (a‐syn) are expressed in the presynaptic terminals of dopaminergic neurons. a‐syn and DAT form a stable heteromeric complex in co‐transfected cells and mesencephalic neurons (Lee, 2001; Wersinger, 2003). Dysregulation of DA homeostasis is implicated in neurodegenerative diseases, drug addiction and neuropsychiatric disorders. a‐syn is suggested to regulate DA homeostasis, but the mechanism of a‐syn modulation of DAT activity is unknown. We examined how a‐syn affects DAT activity by utilizing dual pipette whole‐cell patch clamp recording, and substrate fluorescence imaging, in immortalized DA neurons and cells stably expressing DAT. Co‐expression of a‐syn and DAT decreased the uptake rate of a fluorescent substrate, ASP+, via DAT. Dialysis of 5 or 10 microM a‐syn via the whole‐cell path pipette stimulated a Na+ independent but Cl‐ sensitive inward current in DAT‐expressing cells. This current is absent when heat inactivated a‐syn is dialyzed into the cells. The a‐syn‐stimulated inward current is eliminated by DAT blockers. Our findings indicate that a‐syn stimulates a DAT‐mediated Cl‐ current. Furthermore, our dual pipette whole‐cell patch method may serve as a model to study the effects of a‐syn on DAT function and DA homeostasis.
Development of a substituted phenyldithiolethione as a novel cyclooxygenase inhibitor with minimal gastro‐irritancy
Authors: Jarrott Bevyn , Zanatta Shannon , Williams Spencer John
Published: June 2021

Hydrogen sulfide has been proposed as an endogenous molecule that inhibits early stages of inflammation. Dithiolethiones can be metabolised to hydrogen sulfide and when coupled to diclofenac produce a compound that is an anti‐inflammatory/analgesic agent without the gastrointestinal toxicity of diclofenac (Wallace 2007).

Previous research has shown that compounds containing an antioxidant moiety, 3,5‐di‐tert‐butyl‐4‐hydroxy‐phenyl were cyclooxygenase (COX) inhibitors (Ruiz et al 2003) so we hypothesised that the attachment of a dithiolethione ring onto this antioxidant may give a compound that inhibited COX and also released hydrogen sulfide. We therefore synthesised and tested 5‐(3,5‐di‐tert‐butyl‐4‐hydroxyphenyl)‐3H‐1,2‐dithiole‐3‐thione and this inhibited purified COX‐1 with an IC50 of 2 µM and COX‐2 with an IC50 of 0.25 µM. After oral administration at a dose of 10 µmol/kg, this compound was anti‐inflammatory in a rat model of inflammation and pain (Complete Freund's adjuvant hind paw model) and had reduced gastrointestinal toxicity in rats (n = 9) when compared to an equivalent dose of diclofenac. Also, in view of the lipid solubility of this compound, it may be centrally acting and neuroprotective in diseases with central inflammatory pathologies.
Synchrotron light source imaging of brain tissue shows changes in iron concentration after chronic implantation of electrodes and electrical stimulation
Authors: Lam Ann , Ma Bonita W. , Nichol Helen , Corcoran Michael E.
Published: June 2021

PURPOSE

Various metallic electrodes are used in research and clinical applications because of their purported biological compatibility. However, little is known about the effects on surrounding tissue following subsequent stimulation. Using X‐ray fluorescence imaging, we examined metal distribution in the brain after stimulation with three types of electrodes.

METHODS

Bilateral implantation of stainless steel, nickel‐chromium, or platinum‐iridium electrodes into amygdala was used in a rat preparation of epilepsy. Stage 5 seizures were kindled by a once‐daily application of electrical stimulation. Rapid scanning X‐Ray fluorescence and microprobe analysis of brain sections were performed using the imaging beam lines at the Stanford Synchrotron Radiation Lightsource.

RESULTS

Images showed elevated iron concentrations around the electrode tips in both control and kindled tissue. Concentrations varied according to electrode type and were greater in kindled tissue as compared to yoked controls.

CONCLUSIONS

Chronic implantation of electrodes in the brain results in an increased concentration of iron around the electrode track, which is further increased by stimulation. These findings raise the issue of possible adverse effects from these metal depositions and suggest the need to evaluate the effects of chronic electrical stimulation in clinical settings.

Support: NSERC, CIHR
8,9‐Dihydroxyeicosatrienoic acid does not protect the glomerular filtration barrier
Authors: Sharma Mukut , McCarthy Ellen T , Savin Virginia J , Patel Paresh , Shaw Sean , Falck John R , Imig John D
Published: June 2021

Introduction

Focal segmental glomerulosclerosis (FSGS) leads to end‐stage renal disease. A circulating factor is believed to cause the primary glomerular lesion in FSGS. We have shown that 8,9‐epoxyeicosatrienoic acid (8,9‐EET) protects the glomerular filtration barrier function against the FSGS factor. Presently, we hypothesized that 8,9‐dihydroxyeicosatrienoic acids (8,9‐DiHETrE), vic‐diol of 8,9‐EET produced by soluble (sEH) epoxide hydrolase, is responsible for the observed protective effect of 8,9‐EET.

Methods

We determined the change in glomerular albumin permeability (Palb) in vitro following incubation of rat glomeruli with FSGS‐plasma, FSGS + 8,9‐EET, FSGS + 8,9‐DiHETrE, FSGS + sEH inhibitor AUDA (1‐10 microM) for 15 minutes at 37°C (n=15 in each group). Glomerular sEH protein expression was determined by immunoblotting.

Results

FSGS plasma‐induced increase in Palb (P<0.001 vs. vehicle control) was completely blocked by exogenous 100 nM 8,9‐EET (P<0.001 vs. FSGS) while its metabolite 8,9‐DiHETrE (200 nM) did not cause a significant decrease in Palb (P<0.1 vs. FSGS). AUDA at 1 microM showed a protective effect on Palb against FSGS plasma (P<0.001 vs. FSGS) but 10 microM AUDA did not. Immunoblot analysis showed a relatively weak protein expression of sEH in glomeruli. Summary and

Conclusions

8,9‐EET and AUDA, but not 8,9‐DiHETrE, attenuated the FSGS‐induced increase in Palb. Results suggest that EET metabolism in the glomerulus may be cell‐type specific. We conclude that 8,9‐EET protects against FSGS‐induced glomerular injury and that its vicinal diol 8,9‐DiHETErE does not contribute to the protective effect.
Endogenous Prostacyclin Signaling Regulating MicroRNA Expression in Mammalian Cells
Authors: Mohite Anita J , Chillar Annirudha J , Wijaya Cori , So Shui‐Ping , Gao Xiaolian , Ruan Ke‐He
Published: June 2021

Prostacyclin (PGI2), a key vascular protector and a representative of eicosanoids metabolized from endogenous arachidonic acid (AA), involved in regulating cellular microRNA expression was found. A primary cultured cell line derived from mouse adipose tissue containing the pathway enzymes to metabolize endogenous AA to diverse eicosanoids was transfected with a cDNA of an engineered hybrid synthases which integrated cyclooxygenase‐1 and PGI2 synthase (PGIS) triple catalytic functions. It successfully redirected the cells (PGI2‐cells) stably and dominantly metabolizing the endogenous AA to PGI2. By microarray analysis for 119 miRNAs, expression of miRNA‐711 and 466f‐3p in the PGI2‐cells was dramatically (approximately 15‐folds) upregulated and down regulated, respectively. The up‐ and down‐ regulated miRNAs expression in the PGI2‐cells were completely abolished by specifically inhibiting either the PGI2 signalings through the membrane receptor or the PPARγ pathway. This study provides the evidences that PGI2 could be involved in much broad pathophysiological processes including the controlling of apoptosis, vascular inflammation, atherosclerosis, cancer, implantation, and obesity through its action on up‐ and down‐regulation of the miRNA expressions. The study has created a new dimension to understand the diverse biological functions of eicosanoids derived from endogenous AA in general.
Selective activation of organic nitrates by, and inactivation of, ALDH isoforms
Authors: Page Nathaniel A. , Tsou Pei‐Suen , Beretta Matteo , Mayer Bernd , Fung Ho‐Leung
Published: June 2021

The activation of nitroglycerin (NTG), isosorbide dinitrate (ISDN) and isosorbide‐5‐mononitrate (ISMN) by aldehyde dehydrogenases (ALDH1 and ALDH2), and their subsequent inactivation, were examined. Purified ALDH1 or ALHD2 were incubated with these organic nitrates (ORN), and NO production was monitored by a NO electrode. ALDH2 catalyzed NO production from NTG and ISDN, but not ISMN. In contrast, ALDH1 liberated NO from NTG, ISDN and ISMN similarly (ORN all 1 mM, AUC of NO production in µM*sec: NTG: 35.1± 4.7, ISDN: 30.5±3.1; ISMN: 35.6±1.9). The inactivation of the two ALDH isozymes was examined by incubating each ORN for 15 min, and the ability of these pretreated enzymes to produce NO from 0.1mM NTG was monitored. NTG (0.1 mM) and ISDN (1 mM) completely inactivated ALDH1, while 1 mM ISMN inactivated this enzyme partially (to 34.1±3.7% of control). In contrast, ALDH2 was completely inactivated by 0.1 mM NTG, partially by 1 mM ISDN (to 27.0±23.0% of control), and not by 1 mM ISMN (to 95.0±35.8% of control, p>0.05). The inhibition of ALDH esterase and dehydrogenase activities by ORN showed a similar pattern. These data suggest that activation of ORN to NO by ALDH isoforms, and subsequent enzyme inactivation by ORN, are selective. While NTG and ISDN are both activated by, and in turn inactivated, ALDH1 and ALDH2, ISMN is not a substrate or inactivator of ALDH2, but rather of ALDH1. (Supported in part by NIH grant HL81580).