bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identifying the Secretome and Transmembrane Proteins of Non‐Professional Phagocytes
Authors: Calikyan Anoush , Serizier Sandy , Mondragon Albert , Silvestrini Melissa , Peterson Jeanne , Emili Andrew , Kwan Julian , McCall Kim 
Published: June 2021

Apoptosis and the engulfment of dead cells are essential processes for the proper development and maintenance of an organism. While apoptosis is well‐characterized, many modulatory signals that control apoptotic cell engulfment have not been well described. We use the Drosophila ovary as a model for cell death and clearance, where follicle cells act as non‐professional phagocytes and engulf the dying germline. In this system, several transmembrane receptors, such as Draper, have been found to be essential for nurse cell clearance by follicle cells. However, large‐scale approaches have not been used to determine the signaling molecules of follicle cells that promote cell clearance. We hypothesized that many of the proteins involved in this network of signaling are secreted or are transmembrane proteins. This project, thus, focuses on identifying the “secretome” and transmembrane proteins of engulfing follicle cells by labeling proteins in the endoplasmic reticulum (ER) with a biotin phenol tag. Horseradish peroxidase (HRP) catalyzes the biotinylation of all proximal proteins. When fused to an ER retention signal (KDEL) the expression of HRP is targeted to the ER. We generated flies carrying this HRP‐KDEL construct under UAS control, and then expressed it in desired follicle cell populations. We demonstrated that HRPKDEL localizes around the follicle cell nucleus with immunohistochemistry, suggesting it is expressed appropriately in the endoplasmic reticulum. Moreover, the stains probing for biotinylated proteins and the HRPKDEL construct, respectively, co‐localize, indicative of proximal protein biotinylation. Protein biotinylation was also confirmed by western blot analysis. Ongoing experiments will identify and characterize the biotin‐labeled proteins by mass spectrometry to determine the secretome of follicle cells. By characterizing the secretome of the follicle cell population, we can identify new proteins that regulate the engulfment of apoptotic cells.Support or Funding InformationThe Undergraduate Research Opportunities Program at Boston University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of Hexavalent Chromium on Dictyostelium Chemotaxis and Proteome
Authors: Daniel Hanix Emeral , Moen Rebecca 
Published: June 2021

Hexavalent chromium (Cr6+) is a common environmental pollutant extensively used in industrial processes like chrome‐plating, leather tanning, and paint making that is capable of accumulating in aquatic vascular plants and tissue of animals. The uneven geographical distribution of chromite mining makes this issue a global one and indicates that further study of Cr6+ may lead to targeted treatment in individuals who ingest contaminated water and wildlife or suffer from direct skin contact with contaminated soil. Dictyostelium discoideum is a soil‐dwelling eukaryote that contains many genes that are homologous to human genes while exhibiting a simple lifecycle, restricted cell types and behaviors, and rapid growth. Dictyostelium is widely regarded as a good model organism for studying eukaryotic cell response to toxic compounds. The aim of the study is to determine how hexavalent chromium affects cytoskeletal proteins, specifically actin, and the movement of Dictyostelium in response to Cr6+. A focus is placed on actin and its associated proteins because it has been shown to be a key player specialized stress signaling pathways, and its associated proteins may be multifunctional and may be implicated in a variety of conditions. Chemotaxis, cell movement in response to an external stimulus, was assayed following cell treatment to determine how cell signaling changes as a result of Cr6+ exposure altered the assembly of the actin network. Differences in the actin network of both the treatment and control groups were visualized using fluorescence microscopy. Electrophoresis was then performed on control and treatment groups to resolve proteins and identify specific changes in the expression of actin and its associated proteins.Support or Funding InformationMinnesota State University, Undergraduate Research Center.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Conserved C‐terminal Region of Gα12 and Gα13 Harbors Unique Determinants of SRF Signaling and RhoGEF Binding
Authors: Mull Makenzy L. , Fleming Todd L. , Quick Courtney R. , Meigs Thomas E. 
Published: June 2021

Heterotrimeric G proteins of the G12/13 subfamily play key signaling roles in cell growth, oncogenic transformation, migration, and invasion. The G protein alpha subunits, Gα12 and Gα13, share 67% amino acid identity and several downstream signaling pathways, including transcriptional activation via Serum Response Factor (SRF). Gα12 and Gα13 have multiple, unique binding partners, suggesting divergent signaling mechanisms within this subfamily. To identify different structural features required for growth signaling in Gα12 and Gα13, we utilized amino acid substitutions from non‐SRF signaling invertebrate homologs. Replacing the N‐terminal region of these mammalian α subunits with invertebrate sequence caused no disruption of SRF signaling. Conversely, invertebrate substitution of a highly conserved sequence downstream of the Switch III region abolished SRF signaling by Gα12 and Gα13. In this “Post‐Switch region” of Gα12, a single amino acid substitution disrupted its SRF signaling, while the same mutation in Gα13 had no effect. Replacement of additional residues in this region uncoupled Gα13 from SRF activation, suggesting divergent growth signaling mechanisms in the G12/13 subfamily. In addition, invertebrate substitution in the Post‐Switch region disrupted Gα13 interaction with RhoGEFs, whereas the corresponding SRF‐uncoupled Gα12 retained binding to these effector proteins. To further explore differences in the growth signaling mechanisms of Gα12 and Gα13, we engineered invertebrate chimeras within the C‐terminal α5 helix. These substitutions partially disrupted Gα12‐mediated SRF signaling but were inconsequential for Gα13. Our findings reveal novel C‐terminal motifs in Gα12 and Gα13 that are necessary for their non‐overlapping mechanisms of RhoGEF binding and SRF‐mediated cell growth.Support or Funding InformationWe acknowledge support from the North Carolina GlaxoSmithKline Foundation, the C.D. Spangler Foundation, and the UNC Lineberger Comprehensive Cancer Center.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding G Protein Selectivity of Dopamine Receptors Using Computational Methods
Authors: Portillo Jennifer , Abrol Ravinder , Sakkiah Suguna 
Published: June 2021

Dopamine is important in physiological processes associated with the rewards and pleasure system. Its function is facilitated by five G Protein – Coupled Receptors (GPCRs). The coupling that occurs between the dopamine receptor and G protein dictates the signaling cascade. The dopamine receptors D1 and D5 couple to the Gs protein while the dopamine receptors D2, D3, and D4 couple to the Gi protein. Dysregulation of the dopamine receptors can lead to neuropsychiatric disorders such as schizophrenia. These disorders are difficult to treat due to the side‐effects that transpire. Understanding the coupling that occurs between the dopamine receptors and the G proteins can further assist with the development of pharmaceutical drugs by helping to alleviate some of the side‐effects. Using computational biophysical methods, the coupling between the dopamine receptors and G protein is being studied to understand the structural mechanisms observed in G protein selectivity of these receptors. The dopamine receptor structures for D1 and D2 in complex with the two G proteins (Gs and Gi) have been relaxed in the membrane environment. A structural analysis of those complexes in combination with structural bioinformatics methods has elucidated receptor residues playing a role in the G protein coupling, which has led to the design of receptor mutants with altered G protein selectivities. These mutants are now being characterized by computational biophysical methods before they will be tested in biochemical assays for altered G protein preferences.Support or Funding InformationNational Institutes of Health (NIH) Building Infrastructure Leading to Diversity (BUILD) #5TL4GM118977This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RNA Exclusion Mechanism of the Cytidine Deaminase APOBEC3G
Authors: Solomon William C , Myint Wazo , Hou Shurong , Kanai Tapan , Schiffer Celia A , Matsuo Hiroshi 
Published: June 2021

APOBEC3G (A3G) is a potent antiretroviral protein that specifically targets and deaminates cytidine nucleotides to uracil in single stranded DNA (ssDNA) intermediates produced during HIV replication. A3G displays high selectivity for specific polynucleotide contexts targeting 5′‐CC sites for deamination. A3G binds strongly to RNA via the N‐terminal non‐catalytic domain, which is crucial for recruitment of A3G to the virion, yet A3G displays no deamination activity on the cytidines in single stranded RNA. Our recent structure of A3G bound to ssDNA shows the preferred substrate conformation but does not reveal the mechanisms of substrate selection and non‐substrate exclusion. Here, we report NMR and molecular dynamics (MD) simulation based analysis of the interactions between the C‐terminal catalytic domain of A3G (A3Gctd) and multiple substrate and non‐substrate ssDNAs, which are coupled with deamination assays. The extent of interaction with residues located in helix1 and loop1 (T201‐L220) distinguishes substrate ssDNAs from non‐substrate ssDNAs, revealing the difference between catalytic and non‐catalytic ssDNA‐binding modes of A3Gctd. Using fluorine substituted ribonucleotides we show that 2′‐endo sugar pucker of the target cytidine is an essential factor for effective deamination. Furthermore, MD simulations show that ribose cytidine does not stay at catalytic position during 100ns of simulation time whereas deoxy‐ribose cytidine does. Trajectories of the MD simulation suggest that 2′‐hydroxyl group of RNA forms a hydrogen bond with His257 that chelates Zn2+, which results in loss of two hydrogen bonds between the target cytidine and A3Gctd, dislocating cytosine base from the catalytic position. This study provides mechanistic models of the catalytic selectivity of A3G against RNA.Support or Funding InformationInstitute for Molecular Virology Training Program (NIH grant T32 AI83196) University of MinnesotaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The role of Plasminogen Activator Inhibitor Type‐2 (PAI‐2) in modulating venous thrombus resolution
Authors: Johnson Tierra A , Mukhopadhyay Subhradip , Duru Nadire , Buzza Marguerite S , Antalis Toni M 
Published: June 2021

Deep Vein Thrombosis (DVT) which can arise from vascular endothelial injury, venous stasis, and/or alterations in blood hypercoagulability, affects patients of increased age who have limited to no mobility, paralysis, cancer, surgery and/or trauma. DVT is characterized by thrombi that form on the surface of activated endothelium in both the vein valve pockets and dilated sinuses of the lower limbs and is a highly inflammatory process. Macrophages and neutrophils are known to play key roles, being major producers of inflammatory cytokines, chemokines, and proteases. However, the precise mechanisms and key players that modulate venous thrombosis and its resolution are poorly understood. Plasminogen activator inhibitor type‐2 (PAI‐2) or SerpinB2, a Clade B serine protease inhibitor (SERPIN) was originally identified as an inhibitor of the serine protease, urokinase‐plasminogen activator(uPA) and has also been found to possess unique properties associated with inflammation and cell survival. Utilizing a clinically relevant mouse model of DVT, we discovered that genetic deletion of PAI‐2 accelerates venous thrombus resolution in vivo. PAI‐2 deficiency leads to enhanced venous thrombus resolution, increased proteolytic activity of uPA, and modulation of inflammatory processes. Recent work in our lab has focused on the primary effector cells of thrombus formation and resolution, neutrophils and macrophages respectively. PAI‐2 expression can be found in both resident and activated, neutrophils and macrophages, and may modulate how these cells function on fibrin and collagen matrices found within the thrombus microenvironment. Clarifying the mechanism by which PAI‐2 contributes to venous thrombus resolution has potential to lead to new therapies that accelerate venous thrombus resolution.Support or Funding InformationDepartment of Physiology, Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine and VA Maryland Health Care System, Department of Veterans Affairs, Baltimore, MarylandThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kindlin‐3 recruitment to the plasma membrane in neutrophils precedes high affinity integrin activation
Authors: Wen Lai , Roy Payel , Kiosses William B. , Ginsberg Mark , Ley Klaus 
Published: June 2021

Integrin‐mediated adhesion of circulating leukocytes to the blood vessel wall starts by arrest from rolling. Activation of integrins by inside‐out signaling involves conformational changes from an inactive bent conformation to an extended conformation (E+) with high affinity for ligand binding (H+). The cytoplasmic protein kindlin‐3 is critically required for leukocyte adhesion. Mutations of kindlin‐3 cause leukocyte adhesion deficiency type III (LAD‐III) syndrome, characterized by severe bleeding (a platelet adhesion defect) and recurring infections (a neutrophil adhesion defect). The clinical significance of kindlin‐3 has been extensively studied; however, the molecular mechanism by which kindlin‐3 regulates the β2 integrin activation is unknown. Kindlin‐3 can bind the β2 integrin cytoplasmic tail at a site distinct from the talin‐1 binding site. It remains unclear whether kindlin‐3 affects individual integrin conformation and/or integrin clustering. Here we monitored the spatiotemporal dynamics of kindlin‐3 and β2 integrin conformation changes during neutrophil rolling, arrest and spreading under flow (6 dyn/cm2) using high‐resolution quantitative dynamic footprinting (qDF) microscopy in combination with an integrin conformation‐specific antibody reporting assay and kindlin‐3 fusion reporters. We find that kindlin‐3 is recruited to the plasma membrane during neutrophil adhesion prior to induction of H+ β2 integrin conformation, indicating that kindlin‐3 is required for H+ integrin conformation. Indeed, deletion of kindlin‐3 in neutrophil‐like HL‐60 cells completely abolishes high affinity β2 integrin. Together, our data suggest that kindlin‐3 is permissive for the high affinity β2 integrin conformation.Support or Funding InformationThis work was supported by funding from the National Institutes of Health, USA (NIH, P01‐HL078784).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Testosterone Modulates the Multiplication and Viability of C2C12 Cells Cultivated In Vitro
Authors: Camargo Anne Marielle , Alves Eduardo Donatto , Achcar Jorge Alberto , da Costa Iemma Monica Rosas , Montrezor Luis Henrique 
Published: June 2021

The use of C2C12 cell cultures is strategic for the study of functional mechanisms of skeletal striated muscle, as well as their possible correlations with other tissues and organ systems, in physiological and pathological situations. The objective of this work was to evaluate the effects of different concentrations of testosterone (T) on the multiplication and viability of C2C12 cells, in the absence and presence of flutamide (F), an androgen receptor inhibitor. The cells were cultured (in triplicates) in Dulbecco's Modified Eagle Medium (DMEM) supplemented with fungicide, streptomycin, penicillin, and 2% equine serum, in 48‐well plates (5000 cells / 200 μL‐day zero). The cells were incubated (37 °C, 5% CO2, 95% atmospheric air) for up to 10 days, in the absence (control) and presence of T (10−8 M and 10−4 M, mimicking normo‐ and hyperandrogenemia), and in the absence and presence of F (100 nM). The medium was exchanged every 48 h. Cell counting was performed with a Bio‐Rad TC20™ cell counter. The MTT (3‐ (4,5‐dimethylthiazol‐2‐yl) ‐2,5‐diphenyltetrazolium) reduction method was used to determine cell viability. Statistical analysis of the data employed ANOVA and Fisher's test. Cell multiplication. After 5 days of culture, cell multiplication was inhibited by T at 10−4 M, in the presence of F. After 10 days of culture, the cell count showed inhibition in the presence of T at 10−4 M, in the absence of F, and stimulation in the presence of T at 10−8 M, in the presence of F. Cell viability. After 5 days of culture, cell viability was stimulated by T at 10−8 M, with and without flutamide, and by T at 10−4 M, in the absence of F. After 10 days of culture, cell viability decreased in the presence of T at 10−8 M, without flutamide, and in the presence of T at 10−4 M, with F. The results suggested that the culture time influenced the cellular responses analyzed. In addition, there was a dual effect of T on multiplication and cell viability, in the absence and presence of flutamide, for both culture times studied. Interestingly, when T, in the absence or presence of flutamide, stimulated or inhibited cell multiplication, the cell viability was unaffected by the treatments, and vice versa. The findings suggested that T directed the cellular mechanisms towards multiplication, and sometimes towards metabolic activity. This cell culture model is shown to be feasible for studies of the effects of T mediated by androgen receptors; however, molecular studies are needed in order to obtain a better understanding of the mechanisms involved.Support or Funding InformationFunding: CNPq (Grant 159893/2017‐8)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bioinformatics analysis for heart failure with preserved ejection fraction
Authors: Zhang Wenxi , Huo Yunlong , Tan Wenchang 
Published: June 2021

Heart failure with preserved ejection fraction (HFpEF) represents approximately half of heart failure but little is known about the underlying mechanisms. Dahl salt‐sensitive (Dahl‐SS) rats were fed a high‐salt diet (8% NaCl) from 7 weeks of age to induce HFpEF and controls were fed a normal‐salt diet (0.3% NaCl). Echocardiograms and electrocardiography (ECG) was performed to assess cardiac function. Diastolic dysfunction and preserved ejection fraction along with signs of heart failure was showed and QTc interval on ECG was prolonged (p<0.001) at 14 weeks of age. Hearts were harvested for gene array and label‐free quantitative proteomics. Bioinformatics analysis using DAVID website, GSEA software and BinGO plugin in Cytoscape environment. GO analysis showed microtubule binding and calcium dependent phospholipid binding protein were significant downregulated while oxidative stress response protein were upregulated (p<0.05, q<0.05). GSEA results indicated that gap junction, focal adhesion and actin cytoskeleton regulation gene sets were concentrated in upregulated transcripts (p<0.01, q<0.25) while cardiac muscle contraction and oxidative phosphorylation pathway were downregulated in proteomics (p<0.001, q<0.001). Western blot and PCR experiments showed significant changes in the expression of Dynamin‐1, Integrin‐α9 and MFF, which had important functions including cell stiffness regulation. Our study provides valuable information on the molecular functions and regulatory network of HFpEF, as well as new perspectives on the relationship between diastolic dysfunction and calcium stimulate, cytoskeleton stiffness and oxidative stress.Support or Funding InformationShenzhen Science and Technology Innovation Institution (China) Grant JCYJ20160427170536358 (Y Huo)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The effect of corylin in regulating NLRP3 inflammasome activation in macrophages
Authors: Lai Yi‐Xin , Wu Hsin‐En , Li Chia‐Yang 
Published: June 2021

Chronic inflammation is associated with several diseases and cancers. The activation of inflammasomes has been indicated as a main role in chronic inflammation, which is a group of cytosolic protein complexes, classically consisting an NOD‐like protein (NLR), the adaptor apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC), and caspase‐1. Activation of inflammasome results in assembly of the inflammasome, caspase‐1 activation as well as IL‐1β and IL‐18 secretion. A number of studies have reported that increased concentration of IL‐1β protein in tumor tissues is associated with poor prognosis for cancer patients.Corylin is a main compound isolated from Psoralea corylifloia L., a common Chinese herbal medicine. The Psoralea corylifloia L. has been used in the treatment of skin diseases and osteoporosis. The extracts of this plant has been found to have anti‐oxidative, anti‐bacterial, anti‐diabetic, neuroprotective and immunomodulatory effects. Our previous study demonstrated that corylin inhibits the production of TNF‐α, IL‐6 and NO by murine macrophages through inhibiting the activities of MAPKs and NF‐κB. In the present study, we will examine the effect of corylin in regulating NLRP3 inflammasome activation in macrophages. We used murine macrophages (J774A.1 cells) and human monocytes (THP‐1 cells), and treated with LPS/ATP to induce the activation of NLRP3 inflammasome, then gave different doses of corylin to investigate its effects. The supernatant is used to determine the level of IL‐1β by enzyme‐linked immunosorbent assay (ELISA), and the lysates is used to investigate the level of NLRP3 inflammasome associated protein by Western blot. Our experimental results indicated that corylin inhibits the activation of NLRP3 inflammasome in both murine and human macrophages. We will further examine the effect of corylin on the polarization of macrophages.Support or Funding InformationThis study was supported by the Ministry of Science and Technology, Taiwan, R.O.C. (Grant No. MOST 107‐2320‐B‐037‐019).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.