bluuebunny/crossref_metadata_2025_split huggingface dataset:
PKCθ‐JunB axis via upregulation of VEGFR3 expression mediates VEGFR2‐induced pathological retinal neovascularization
Authors: Kumar Raj , Mani Arul M. , Singh Nikhlesh K. , Rao Gadiparthi N. 
Published: April 2020

Pathological retinal neovascularization is the most common cause of vision loss. PKCθ has been shown to play a role in type 2 diabetes, that is linked to retinal neovascularization. Based on these findings, in this study, we have determined the role of PKCθ in retinal neovascularization using human retinal microvascular endothelial cells (HRMVECs) and oxygen‐induced retinopathy (OIR) as models. VEGFA induced PKCθ phosphorylation in a time dependent manner in HRMVECs and down regulation of its levels attenuated VEGFA‐induced HRMVECs migration, sprouting and tube formation but not DNA synthesis. Furthermore, genetic deletion of PKCθ in C57BL/6 mice inhibited hypoxia‐induced retinal endothelial cell proliferation, tip cell formation, sprouting and neovascularization. We also observed that VEGFA induces VEGFR3 expression via PKCθ‐dependent JunB induction in the regulation of HRMVEC migration, sprouting, and tube formation in vitro and OIR‐induced retinal endothelial cell proliferation, tip cell formation, sprouting and neovascularization in vivo. In addition, PKCθ‐JunB axis modulates VEGFA‐induced VEGFR3 promoter activity and its expression downstream to VEGFR2 activation both in vitro and in vivo. Depletion of VEGFR2 or 3 levels attenuated VEGFA‐induced HRMVEC migration, sprouting and tube formation in vitro and retinal angiogenesis in vivo and it appears that these events were dependent on STAT3 activation. In addition, VEGFR3 appears to be mediating retinal neovascularization in a ligand dependent and independent manner downstream to VEGFR2 activation. Together, these observations infer that PKCθ‐dependent JunB‐mediated VEGFR3 expression targeting STAT3 activation is required for VEGFA‐VEGFR2 induced pathological retinal neovascularization.Support or Funding InformationThis work was supported by a grant EY04856 from NIH to GNR.
Deleted in Colorectal Cancer Inhibits Protein Synthesis, But How?
Authors: Spear Elizabeth A. , Filbin-Wong Megan E. 
Published: April 2020

Deleted in colorectal cancer (DCC) is a chemotropic transmembrane receptor known to regulate neuron growth by stimulating or suppressing protein synthesis machinery. DCC does this by sensing an extracellular signal known as netrin‐1. In the absence of netrin‐1, DCC tethers translation machinery with its cytoplasmic tail (C‐tail), blocking ribosome function. In the presence of netrin‐1, DCC homodimerizes, releasing the translation machinery for local protein synthesis. While netrin‐1’s role is understood, the C‐tail‐translational machinery complex is poorly defined. Our lab focuses on which portion of the DCC C‐tail is necessary and sufficient at regulating protein synthesis. Using site‐directed mutagenesis, traditional cloning, and recombinant protein expression we have created a series of C‐tail truncation mutants. We measured the inhibitory function of each mutant using an in vitro luciferase assay. Our results indicate that the inhibitory region of the tail lies toward the N‐terminus. By defining the inhibitory portion of the C‐tail we have developed a model for how it is interacting with the translation machinery. Collectively, our data contribute to a growing body of knowledge about the relationship between chemotropism and protein synthesis.Support or Funding InformationResearch reported in this poster was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM134393. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Support was also provided by an undergraduate research grant to E.A Spear from the Metropolitan State University of Denver Undergraduate Research Program.
Tracking mTORC1 Activity in the Nucleus: Distinct Regulation Revealed by Locationspecific Inhibition of Akt
Authors: Zhou Xin , Zhong Yanghao , Kunkel Maya T. , Newton Alexandra C. , Zhang Jin 
Published: April 2020

The mechanistic target of rapamycin complex 1 (mTORC1) is a kinase complex that contains the protein kinase mTOR, the key scaffolding protein Raptor, and several other core components. It is known to sense diverse upstream signals, such as growth factors, nutrient and energy status. When activated, mTORC1 integrates these signals to critically regulate distinct cellular processes, including cell growth, metabolism, and autophagy. An intriguing question is how mTORC1 is spatially regulated to exert widespread controls on downstream effectors. Besides the well studied mTORC1 activation at the lysosome, the regulation of this complex within other subcellular compartments is less clear. Previously, we showed that growth factor induces nuclear mTORC1 activity by utilizing a genetically encoded mTORC1 activity reporter targeted to the nucleus, providing initial evidence for active mTORC1 in the nucleus. Here we probe the regulation of growth factor‐induced nuclear mTORC1 activity by combining location‐selective kinase inhibition, live‐cell imaging and cellular biochemistry. We developed Akt Substrate‐based Tandem Occupancy Peptide Sponge (Akt‐STOPS) that allows location‐specific perturbation of Akt, a critical upstream kinase, at distinct subcellular compartments. Expression of Akt‐STOPS in the nucleus largely suppressed the growth factor stimulated Akt activity without significant inhibition of cytosolic Akt activity. Using Akt‐STOPS, we showed that growth factor‐stimulated nuclear mTORC1 activity requires nuclear Akt activity. Furthermore, we showed that growth factor stimulation induces nuclear translocation of Raptor, which is sensitive to importin inhibition, and that Raptor forms a complex with mTOR in the nucleus . The nuclear translocation of Raptor specifically depends on the nuclear Akt activity, as expression of nuclear Akt‐STOPS significantly lowered the nuclear translocation of Raptor and reduced the phosphorylation of RanBP, a critical regulator of Ran GTPase. Lastly, localizing exogenous Raptor to the nucleus in Raptor knock‐down cells rescues nuclear mTORC1 activity. Taken together, these results reveal a new mode of dynamic regulation of mTORC1 by recruiting a critical scaffolding protein, distinct from the mode of lysosomal mTORC1 activation. Diversity in the regulation of mTORC1 could lead to specificity in functional controls and also provide opportunities for selective targeting.Support or Funding InformationThis work was supported by NIH grants, R35 CA197622 and R01 GM111665 to J.Z., and NS047101 to UCSD Microscopy Core.
ADGRF1 signaling pathways in Breast Cancer
Authors: Abdulkareem Noor Mazin , Bhat Raksha , Qin Lanfang , Schiff Rachel , Trivedi Meghana V. 
Published: April 2020

BackgroundAdhesion G‐protein‐coupled receptors (GPCRs) form the second largest GPCR subfamily. They control many cellular processes involved in cancer, such as cell adhesion, invasion/migration, and stem cell function. Most of adhesion GPCRs are still orphans without any known endogenous ligands. In breast cancer, ADGRF1, previously called GPR110, is overexpressed in 2 aggressive subtypes of breast cancers, HER2+ and triple‐negative. We have found that HER2+ cells with ADGRF1 overexpression in doxycycline‐inducible manner formed more secondary mammospheres and more colonies in the soft agar assay and had higher % of Aldefluor+ cell population. However, ADGRF1‐overexpressing BT474 cells formed fewer tumors in mice and had reduced tumor growth compared to control.AimTo understand the downstream signaling of ADGRF1 that may contribute to the opposing findings in vitro and in vivo.MethodsThe experiments were conducted using two HER2+ breast cancer cell lines: BT474 and SKBR3 stable cells overexpressing ADGRF1 in doxycycline‐inducible manner and MDA‐MB‐231, a triple‐negative breast cancer cell line with endogenous ADGRF1 overexpression. The coupling of ADGRF1 to Gαs and Gαq pathway was assessed by measuring cAMP (AlphaScreen assay) and IP1 (Homogeneous Time‐Resolved Fluorescence assay), respectively and by co‐immunoprecipitation and immunoblotting. The effect of synaptamide, an ADGRF1 agonist, was also assessed on cAMP and IP1. The effects of Gαs or Gαq pathway activators were assessed on the potential of mammosphere formation (using Mammosphere assay).ResultsThe basal levels of cAMP and IP1 was significantly higher in doxycycline‐treated (+Dox) BT474 cells overexpressing ADGRF1 compared to cells without doxycycline (−Dox). Synaptamide (1–100 nM) did not further increase cAMP or IP1 in these cells. On the other hand, synaptamide increased cAMP but not IP1 in a concentration‐dependent manner in MDA‐MB‐231 cells. Immunoprecipitation with both anti‐Gαs and anti‐Gαq antibodies pulled down ADGRF1 in +Dox but not −Dox treated BT474 and SKBR3 cells. Forskolin, an activator of adenylyl cyclase that increases cAMP, reduced mammosphere formation in MDA‐MB‐231 cells. The effect of Gαs or Gαq pathway activators on mammosphere formation in BT474 and SKBR3 clones is being assessed in ongoing experiments.ConclusionOur results show that ADGRF1 couples to both Gαs and Gαq pathways. However, our data do not indicate that differential coupling leads to opposing in vitro versus in vivo effects. Additional studies with pathway activators as well as acute versus chronic activation of ADGRF1 may elucidate ADGRF1 pathways relevant in breast cancer biology.Support or Funding InformationDepartment of Defense Grants W81XWH‐14‐1‐0340 and W81XWH‐14‐1‐0341 to Drs. Trivedi and Schiff, respectively.
Animals with Endometriosis Experience Beneficial Effects and Localized Modulation of Cytokine Levels when Exposed to Voluntary Wheel Running
Authors: Cruz Myrella L , Velazquez-Cruz Johnathan , Chompre Gladys , Rivera Luis A , Reyes-Bonilla Merie M , Appleyard Caroline B 
Published: April 2020

A physically active lifestyle has been shown to confer preventive health benefits in many chronic conditions, in part by enhancing immunological health. Prolonged moderate exercise increases secretion of IL‐6 that may also promote secretion of the anti‐inflammatory cytokine IL‐10. Endometriosis is a chronic gynecological disorder characterized by peritoneal inflammation, chronic pelvic pain, fibrosis, and growth of endometrial tissue outside the endometrial cavity. We recently demonstrated protective effects of prior exercise in this condition, however it is still unclear whether physical activity can alleviate endometriosis that has already developed.HypothesisVoluntary physical exercise can promote immune strength helping to reduce symptoms in animals with endometriosis.MethodsEndometriosis was induced in female Sprague Dawley Rats by implanting four pieces of uterine tissue next to the intestinal mesentery on day 0, while Sham controls received sutures only. Endo (n=11) and Sham controls (Sham; n=11) received no exercise. Another group of endometriosis (EEX; n=10) and sham animals (SHEX; n=10) had access to a running wheel after induction of endometriosis until time of sacrifice at day 60. At sacrifice the developed vesicles, colon, serum and peritoneal fluid were collected and stored for future analysis.ResultsEndo animals developed vesicles in 79.55% of the implants with an average total area of 71.02±15.50mm2 and average developed vesicle weight of 0.12±0.04g. No vesicles developed in Sham or SHEX. Endo had significantly increased macroscopic colonic damage (p<0.0001 vs Sham and SHEX). Exposure to exercise significantly decreased the number of vesicles that developed (35.00%, p<0.01), as well as their size (10.23±4.24mm2, p<0.01), and weight (0.03±0.01g, p<0.05). EEX had significantly less macroscopic (p<0.0001) and microscopic (p<0.05) colonic damage compared to Endo. Exercise increased the release of various cytokines and chemokines including MIP1 (p<0.01), IL‐10, IP10 (p<0.05), RANTES, and IL‐6 (p<0.05) in peritoneal fluid compared to Endo. Changes in cytokine levels at the systemic level were less pronounced.ConclusionsOur results suggest that voluntary physical activity might alleviate endometriosis associated progression of symptomatology, and enhance immune competency by impacting various inflammatory cytokines and angiogenic factors. Taken together, this opens the potential for further exploration of voluntary exercise as a complementary therapy in the patient population for whom current treatments are still non‐curative with many side effects.Support or Funding InformationSupported in part by R15AT009915, R25GM082406, U54MD007579.
Loss‐ and Gain‐of‐Function Mutations in the Type 1 IP3 Receptor (IP3R1) Affect Myogenic Constriction of Cerebral Resistance Arteries in Response to Intraluminal Pressure
Authors: Zhu Hai-Lei , Walsh Emma , Sutherland Cindy , Chen S.R. Wayne , Braun Andrew , Cole William C. 
Published: April 2020

The constriction and relaxation of resistance arteries/arterioles in response to changes in intraluminal pressure, referred to as the myogenic response, is a key physiological mechanism for the regulation of blood pressure and flow. Despite the recognized contribution of phospholipase C activity and IP3‐mediated intracellular Ca2+ release to the myogenic response, a lack of selective pharmacological agents to target the predominant IP3R1 Ca2+ release channel has prevented interrogation of this key mechanism. Mutations in the IP3R1 are known to produce either loss‐of‐function (LOF) or gain‐of‐function (GOF) phenotypes in humans that impact cerebellar activity (e.g. ataxia), yet the effect of these mutations on the cardiovascular system is not established. Here, we employed a novel genetic knock‐in mouse model with a single point mutation in the transmembrane S6 helix of IP3R1 that confers either a LOF (D2594A) or GOF (D2594K) phenotype. In preliminary experiments, the LOF and GOF mutations shifted IP3‐mediated ER Ca2+ release ~2‐fold to the right and left, respectively, compared to wild‐type (WT) IP3R1 in permeabilized HEK293 cells. In endothelium‐denuded posterior cerebral arteries of 3–4 month old WT mice, myogenic vasoconstriction was evident at ≥40 mmHg, maximal at ~120 mmHg, and declined at >120 mmHg (n=7). In contrast, myogenic constriction was initiated at a higher pressure (≥50 mmHg) and was smaller in amplitude in arteries of heterozygous IP3R1 LOF mice (n=8). In IP3R1 GOF vessels (n=7), myogenic tone developed at a lower pressure (≥20 mmHg), was maximal at ~80 mmHg, and declined at >90 mmHg. Relative levels of phosphorylation of myosin regulatory light chain (LC20) and myosin phosphatase targeting subunit (MYPT1) at 10, 40, 80 and 120 mmHg, and of globular (G‐) actin content at 10, 60 and 120 mmHg, in arteries from WT, LOF and GOF mice were assessed by ultra‐high sensitivity immunoblotting. An identical pressure‐dependent increase in phospho‐LC20 content was detected in WT, LOF and GOF vessels (n=5 each). Although phospho‐MYPT1‐T855 content increased with intraluminal pressure in arteries of WT and LOF mice, it did not change in vessels of GOF mice. Elevation of intraluminal pressure decreased in G‐actin content in arteries of WT mice, did not affect G‐actin content in LOF vessels, and caused a significantly greater decrease in GOF arteries. Taken together, these data demonstrate that genetic modification of IP3R1‐mediated Ca2+ mobilization affects the myogenic response of cerebral arteries to intraluminal pressure, a vascular impairment that may contribute to neurological dysfunction in patients with LOF and GOF IP3R1 mutations.Support or Funding InformationThis work was funded by the following research grants from the Canadian Institutes of Health Research: MOP‐97988 (WCC) MOP‐142467 (APB) and PJT‐155963 (APB, WC and WCC).
Sex differences in the activation of renal cell death pathways in diabetic vascular endothelial cell ET‐1 knockout mice
Authors: De Miguel Carmen , Martinez Vianna G. , Almutlaq Rawan , Pollock David M. , Pollock Jennifer S. 
Published: April 2020

Contrary to other renal diseases that progress faster in males than females, diabetic kidney disease advances at a similar rate in both sexes. The role of endothelium‐derived ET‐1 in the development of diabetic nephropathy remains unclear. These studies were designed to examine the role of vascular ET‐1 in diabetic kidney disease, and to determine if it plays a role in the loss of renal protection in diabetic females. Hyperglycemia was induced in male and female vascular endothelial cell ET‐1 knockout (VEET KO) and floxed ET‐1 mice with streptozotocin (STZ, 50 mg/kg i.p., 5 consecutive days). 10 weeks later, urine and kidneys were collected and kidney damage and cortical expression of cell death pathways were assessed. In response to diabetes, female VEET KO mice displayed greater interstitial fibrosis and cortical tubule dilation. Glomerulosclerosis, GFR, NGAL excretion and nephrin excretion were not different between sexes or genotypes. Interestingly, lack of vascular ET‐1 led to decreased albumin excretion with diabetes, although no sex difference was found (VEET KO vs. floxed ET‐1: 51.6 ± 4.3 vs. 104.1 ± 3.3 mg/day, p<0.05; n=5–7/group). We also found a sex effect in protein and KIM‐1 excretions, with greater excretion of these parameters in females than males (females vs. males, protein: 3.8 ± 0.6 vs. 1.8 ± 0.1 mg/day; KIM‐1: 3.5 ± 0.0 vs. 1.6 ± 0.5 pg/day; p<0.05; n=5–7/group). Compared to diabetic male VEET KO mice, absence of vascular ET‐1 in females resulted in the cortical upregulation of 48 genes involved in cell death pathways (16 genes each for autophagy, necrosis and pro‐apoptosis; 2–4 fold increase, p<0.05, n=3/group). These results highlight the protective role that vascular ET‐1 plays in females, but not males, against the development of diabetic nephropathy.Support or Funding InformationFunded by NIH K01HL145324 and UAB Diabetes Research Center Pilot Project grant to CDM, and UAB KURE R25 DK115353 to VGM and JSP.
Biophysical Studies of a Protein of Unknown Function from Cancer‐Associated Murine Herpesvirus‐68
Authors: Kloehn Jaime C. , Gorres Kelly 
Published: April 2020

Members of the Herpesvirus family cause several types of diseases to its mammalian hosts, including infectious mononucleosis, Hodgkin’s lymphoma, and primary effusion lymphomas. Herpesviruses are characterized by their ability to replicate and spread infection in the active lytic phase, but also maintain themselves within a host during the inactive latent phase. Within the gammaherpesvirus subfamily, a conserved protein of unknown function, with a highly conserved domain of unknown function, is expressed during the lytic phase. The protein was observed to be essential to viral replication during the lytic phase of infection when the gene was knocked out of the murine gammaherpesvirus‐68 (MHV68). To characterize its biophysical properties, the protein of unknown function from MHV68 was purified. Analysis of the protein through Circular Dichroism spectroscopy determined the secondary structure to be predominantly alpha‐helical. The thermal denaturation of the protein was pH dependent. Additionally, using a high throughput screen, the thermal stability of the protein was increased in the presence of small organic acids. The goal of the biophysical and structural analyses is to identify the function of the protein in the viral life cycle and to determine if the protein can be targeted to inhibit replication of gammaherpesviruses and their associated diseases.Support or Funding InformationThis work was supported by UWL College of Science and Health, UWL Undergraduate Research and Creativity, and Hardy W. Chan and Sons Chemistry Summer Undergraduate Research Fellowship.
Evolution of Post‐natal Mouse Respiratory Physiology Revealed by Machine Learning‐based Data Analytics
Authors: Wang Wesley , Alzate-Correa Diego , Alves Michele Joana , Jones Mikayla , Garcia Alfredo , Czeisler Catherine Miriam , Otero José Javier 
Published: April 2020

Current methods to analyze respiratory behavior utilize plethysmography to acquire standing values of frequency, volume and flow at specific time points in murine maturation. However, these factors have been independently analyzed as a function of time with no further analysis of the interplay these variables have amongst each other in the context of maturation. We have developed a R‐based analytical workflow to examine how variation and relationships of frequency (f) and tidal volume (TV) with respect to inspiratory and expiratory parameters shift with age in mice. Our analysis evidences that deeper physiological relationships and variation in breathing behavior can be seen with age. Moreover, models with respect to age are predictive‐‐suggesting a statistical method to evidence changes in respiratory breathing interplay with age. To further this, we designed learning algorithms in R to test the ability to predict age based on these parameters. This data indicates that respiratory pattern control changes and strengthens with age and provides new insight to model paradigms of respiratory maturation in developmental research.Support or Funding InformationThis work was supported by NIH/NHLBI (R01HL132355 for CMC, JJO).