bluuebunny/crossref_metadata_2025 huggingface dataset:
A Physical Model Overlay of Wild Type Influenza Virus A Neuraminidase and the His274Tyr Mutant Reveals the Basis of Drug Resistance and Catalysis
Authors: Manzaro Abigaile Grace , Blanchard Allison , Smith Travon , Wilkins Taylor , Guzman Karen
Published: April 2020

The influenza virus is the causative agent of the flu, a contagious respiratory illness that causes morbidity, loss of productivity, and, in some instances, death. Although the seasonal flu is typically only life threatening to high‐risk groups, exceptionally virulent forms of the virus that emerge during pandemics, can even threaten strong and otherwise healthy individuals. A strong arsenal of antiviral drugs is essential during a pandemic, but drug resistance threatens our preparedness. A key target for antivirals is neuraminidase, an enzyme that cleaves glycosidic bonds between sialic acid residues on cell surface glycoproteins so that the virus can infect or escape from host cells to repeat the cycle of infection. The enzyme exists as a homo‐tetramer with calcium ions stabilizing the protein structure. Antiviral medications, including Oseltamivir and Zanamivir, inhibit neuraminidase activity but resistance to Oseltamivir is most prevalent. The challenges posed by the rapid evolution of drug resistance have led Dr. Schiffer and her colleagues to develop more innovative ways, based on structural analyses to formulate drugs that are immune to resistance. We chose to focus on resistance of the His274Tyr neuraminidase mutant to the drug Oseltamivir using the crystal structure solved by Collins and colleagues that demonstrates that this resistance is due to a shift in the position of Glu276 into the hydrophobic pocket of the active site. Using the software application, Jmol, we designed physical models based on the overlaid crystal structures of the wild type and the His274Tyr mutant to visualize the shift in Glu276 and its impact on binding of each of three potential ligands: Oseltamivir, Zanamivir, and Sialic Acid. In addition, the catalytic residues Tyr406 and Glu277 were displayed to visualize the lack of impact on catalytic cleavage of the natural substrate, but continued inhibition by Zanamivir. By designing a physical model of Neuraminidase to emphasize the dimensional and catalytic residues, a better understanding of the protein’s function and potential inhibition by drugs can be obtained and may lead to a more effective way to design drugs in the future.

Support or Funding Information

Support for the CREST Project is through NSF‐IUSE #1725940.
Evidence that Dopamine is the Physiological Agonist of Spinal Ghrelin Receptors and Acts Through Heteromeric Dopamine D2/ Ghrelin Receptors
Authors: Furness Sebastian G.B. , Pustovit Ruslan V. , Ringuet Mitchell T. , Molero Juan C. , Fothergill Linda J. , Furness John B.
Published: April 2020

Ghrelin receptors (GhrR) occur in autonomic control centers in the spinal cord. However, the only natural activator of GhrR, ghrelin, is absent from the spinal cord. Therefore, we sought another explanation for the conservation of spinal GhrR across species and the consistency of stimulatory effects of GhrR agonists on autonomic centers in the spinal cord.

We found that agonists of dopamine 2 receptors (DRD2) applied to spinal defecation centers in the rat mimic actions of ghrelin receptor agonists to stimulate colorectal propulsion and defecation. Here we present data indicating that the effects through spinal GhrR receptors are mediated by dopamine acting at DRD2 that is coupled to GhrR. In rats, we activated the defecation pathways by a natural stimulus, water avoidance stress, which caused increased fecal output in the rats. The responses were inhibited both by the DRD2 antagonist, sulpiride, and by the GhrR antagonist, YIL781, applied systemically. The stimulation of colorectal propulsion by the DRD2 agonist, quinpirole, applied directly to the defecation centers, was also reduced by GhrR antagonism with YIL781 (systemic). Colorectal effects of DRD2 and GhrR agonists were prevented by cutting the pelvic nerves, indicating that their sites of action are in the CNS. We used RNAScope to localize sites of DRD2 and GhrR expression in the spinal cord. DRD2 and GhrR mRNA were colocalized in autonomic preganglionic neurons, identified by their expression of choline acetyltransferase, at the levels of the defecation centers. Dual localization of the receptors occurred in the majority of ChAT neurons in both the intermediolateral (IML) and intermediomedial (IMM) autonomic cell groups. ChAT, DRD2 and GhrR mRNAs were expressed at similar levels in the neurons. In HEK cells transfected with DRD2, dopamine receptor agonists inhibited adenylyl cyclase but had no effect on cytoplasmic Ca2+. GhrR agonists had no effect in DRD2 cells. In cells transfected with DRD2 plus GhrR, agonists of either receptor evoked increases in cytoplasmic Ca2+. The effects were antagonized by the GhrR antagonist, YIL781, in both cases. We conclude that the involvement of spinal GhrR in colorectal control in the absence of ghrelin, that has been demonstrated both in human and in animal models, is explained by dopamine being the transmitter that acts at DRD2/ GhrR heteromeric receptors

Support or Funding Information

Florey Institute of Neuroscience and mental health
Fusogenic liposomes effectively deliver resveratrol to the cerebral microcirculation and improve endothelium‐dependent neurovascular coupling responses in aged mice.
Authors: Wiedenhoeft Tabea , Tarantini Stefano , Nyúl-Tóth Ádam , Csipo Tamas , Balasubramanian Priya , Yabluchanskiy Andriy , Lipecz Agnes , Kiss Tamas , Csiszar Anna , Csiszar Agnes , Ungvari Zoltan
Published: April 2020

Adjustment of cerebral blood flow (CBF) to the increased oxygen and nutrient demands of active brain regions via neurovascular coupling (NVC) has an essential role in maintenance of healthy cognitive function. In advanced age, cerebromicrovascular oxidative stress and endothelial dysfunction impair neurovascular coupling, contributing to age‐related cognitive decline. Recently we developed a resveratrol (3,4′,5‐trihydroxystilbene)‐containing fusogenic liposome (FL‐RSV)‐based molecular delivery system that can effectively target cultured cerebromicrovascular endothelial cells, attenuating age‐related oxidative stress. To assess the cerebromicrovascular protective effects of FL‐RSV in vivo, aged (24‐month‐old) C57BL/6 mice were treated with FL‐RSV for four days. To demonstrate effective cellular uptake of FL‐RSV, accumulation of the lipophilic tracer dyes in cells of the neurovascular unit was confirmed using two‐photon imaging (through a chronic cranial window). NVC was assessed by measuring CBF responses (laser speckle contrast imaging) evoked by contralateral whisker stimulation. We found that NVC responses were significantly impaired in aged mice. Treatment with FL‐RSV significantly improved NVC responses by increasing NO‐mediated vasodilation. These findings are paralleled by the protective effects of FL‐RSV on endothelium‐dependent relaxation in the aorta. Thus, treatment with FL‐RSV rescues endothelial function and NVC responses in aged mice. We propose that resveratrol containing fusogenic liposomes could also be used for combined delivery of various anti‐geronic factors, including proteins, small molecules, DNA vectors and mRNAs targeting key pathways involved in microvascular aging and neurovascular dysfunction for the prevention/treatment of age‐related cerebromicrovascular pathologies and development of vascular cognitive impairment (VCI) in aging.

Support or Funding Information

This work was supported by grants the Oklahoma Center for the Advancement of Science and Technology, the Oklahoma Shared Clinical and Translational Resources (OSCTR) program funded by the National Institute of General Medical Sciences (GM104938), the NIA‐supported Geroscience Training Program in Oklahoma (T32AG052363), the Oklahoma Nathan Shock Center (P30AG050911), the Cellular and Molecular GeroScience CoBRE (1P20GM125528, sub#5337), and the NIGMS supported Center of Biomedical Research Excellence (CoBRE)..
The inflammatory cytokine, interleukin‐1‐beta, drives central immune cell infiltration, changes in sympathetic nerve activity and blood pressure
Authors: Yao Song Tieng , Chan Alicia Lai Ting , Elsaafien Khalid , Korim Willian Seiji
Published: April 2020

Introduction

The area postrema (AP) is a brain region that lacks a blood brain barrier allowing blood‐borne factors to access the brain. Since the AP is connected to numerous brain regions that play a key role blood pressure regulation, we hypothesised that blood‐borne inflammatory factors such as interleukin‐1 beta (IL‐1β) might play a role in the development of hypertension. In support of this hypothesis, plasma Interleukin‐1 beta is increased in hypertensive patients (Dalekos et al., 1997, J Lab Clin Med,129: 300–8). Furthermore, the receptors for IL‐1β are abundantly expressed in the AP. Here we test whether IL‐1β can act on the AP to cause an increase in blood pressure and renal sympathetic nerve activity (RSNA) in rats.

Methods

Blood pressure and renal sympathetic nerve activity (RSNA) were recorded and analyzed after microinjection of different doses of IL‐1β into the AP (10ng and 25ng) of anaesthetised rats. In order to show that IL‐1β, specifically causes an increase in blood pressure, specific IL‐1 receptor blocking antibodies (IL‐1Ra) were injected into the AP prior to a 25ng IL‐1β injection. The AP was extracted for fluorescent activated cell sorting (FACS).

Results

Experiments showed that microinjection of 25ng IL‐1β caused a significant (p<0.01; Mann‐Whitney test) increase in both systolic blood pressure (19.6 ± 3.6 mmHg) and heart rate (55 ±10 bpm) compared with control injections of saline (SBP: 5.47 ± 1.58; HR 12 ± 6). Pre‐treatment with IL‐1Ra attenuated the response to IL‐1β, and was comparable with saline controls. IL‐1β also caused an increase in RSNA (137% ± 43%) and FACS revealed an increase in immune cell infiltration. Immunohistochemistry also revealed that IL‐1β injection results in Fos activation in the AP.

Conclusions

IL‐1β injection appears to activate neurons in the area postrema which leads to an increase in blood pressure. By providing new insight into the actions of IL‐1β in the brain, we show that IL‐1β might be a potential target for future treatments for hypertension.

Support or Funding Information

Australian Research Council (FT170100363)National Health and Medical Research Council (GNT 1079680)Rebecca L Cooper Medical Research Foundation
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 Information

This 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 Information

Research 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 Information

This 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

Background

Adhesion 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.

Aim

To understand the downstream signaling of ADGRF1 that may contribute to the opposing findings in vitro and in vivo.

Methods

The 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).

Results

The 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.

Conclusion

Our 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 Information

Department 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.

Hypothesis

Voluntary physical exercise can promote immune strength helping to reduce symptoms in animals with endometriosis.

Methods

Endometriosis 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.

Results

Endo 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.

Conclusions

Our 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 Information

Supported 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 Information

This 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 Information

Funded 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 Information

This 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 Information

This work was supported by NIH/NHLBI (R01HL132355 for CMC, JJO).
RNA‐Protein Interactions Involved in Persistence of Cytomegalovirus
Authors: Lee Jessica Erin , Kulesza Caroline
Published: April 2020

Human Cytomegalovirus (HCMV) is a ubiquitous herpes virus and usually asymptomatic among healthy individuals. Congenital HCMV infections are the leading cause of virally induced birth defects and HCMV causes severe disease in immunocompromised patients. Noncoding RNA is a class of RNA molecules that have a wide range of structures and functions which can exhibit cell‐specified specialization and expression. HCMV encodes many ncRNAs that range in size from small (miRNAs) to large stable introns. HCMV encodes several long noncoding RNAs (lncRNAs) of various function. Previous work has shown that HCMV encodes a lncRNA called RNA5.0. The ortholog of RNA5.0 in the MCVM, RNA7.2, has shown to play a role in virus persistence within host salivary glands. However, the molecular interactions/mechanism of this process remains unclear. In order to begin characterizing the molecular function of RNA5.0, we will use the RaPID system to screen for RNA‐protein interactions. The RaPID system recruits a highly active biotin ligase to tag proteins bound on the RNA. Previous work demonstrated that the 3′ end of RNA5.0 is related to the processing and stability of the RNA. There is a hairpin loop that is absolutely required for RNA stability. Several different length fragments spanning the hairpin loop have been cloned into the RaPID system. These constructs will be used to purify RNA binding proteins. Mass spectrometry analysis will be used to identify specific HCMV RNA binding proteins. The identified binding proteins might point to specific pathways involved in viral persistence that could be targeted for potential treatment for CMV.
Biochemical Evaluation of Homeopathic Remedies as Over the Counter Treatments for Vaginal Yeast Infections
Authors: Napoleon Sierra , Cole Marsha
Published: April 2020

A growing number of women around the world are turning to homeopathic remedies to cure vaginal yeast infections. Along with natural suppositories and yeast arrests, a new social media driven product known as Yoni PearlsTMare becoming an online sensation. While many women are taking to social media to give testaments to the product’s efficiency, very little research has been done on the usage, effectiveness, and safety of using these Yoni PearlsTMas well as other homeopathic options. To attempt to decipher this trend, yeast arrest, Yoni Pearls, boric acid suppositories, and tea tree oil suppositories were placed in fermentations performed on Saccharomyces cerevisiaein simulated vaginal fluid. The first major finding seen was the difference in yeast growth for each media. The yeast was inhibited more in the simulated vaginal fluid than when in only an aqueous glucose (5g/L) solution. This difference was attributed to chemical inhibitors in the simulated vaginal fluid. When the effect of the homeopathic treatments were studied in the aqueous glucose solution, significant (P<0.5) differences were observed in the amount of CO2produced. Fermentation yields were measured stoichiometrically as CaCO3by capturing the CO2 in a Ca(OH)2 saturated solution. The homeopathic treatments decreased the fermentation yields in this order: Tea tree oil suppository, Yoni PearlTM, Yeast Arrest, boric acid suppository. Further directions include determining which chemicals in the stimulated vaginal fluid are inhibiting S.cerevisiae fermentation, replicating the experiments on Candida albicans, and using Bovine Serum Albumin assay to determine anti‐inflammatory characteristics.
Kir7.1 Disease Mutation in the Inner Pore is Critical for K+ Permeation
Authors: Beverley Katie Marie , Steffen Jack Thomas , Heyrman Joseph Andrew , Shahi Pawan Kumar , Pattnaik Bikash Ranjan
Published: April 2020

Background

Inwardly rectifying potassium (K+) channels (Kir) are critical for maintaining membrane potential and K+ homeostasis across many tissues. Mutations in the KCNJ13 gene that encodes for Kir7.1 protein, located in the retinal pigmented epithelium (RPE), contribute to the development of pediatric blindness. One such point mutation in the KCNJ13 gene c.458C>T changes Threonine (T) to Isoleucine (I) at amino acid position 153. T153 is located within the second transmembrane domain, lining the inner pore of the tetrameric protein.

Hypothesis

We hypothesized that T153I would not affect protein translation, assembly, or trafficking. Because of the specific location of T153 within the pore, the mutation might affect ion permeation through the channel.

Methods

HEK293 cells in culture were transfected with either GFP tagged wildtype (WT) Kir7.1 or T153I plasmid DNA. Protein from these cells was isolated and used for Western blotting and Mass‐spectroscopy. Confocal live‐cell imaging, using Nikon‐C2, mapped the expression of GFP fusion protein. Hoescht marked the nucleus, and WGA‐Alexa 594 labeled the plasma membrane. Images were subjected to off‐line analysis with NIS Elements. Whole‐cell patch‐clamp electrophysiology provided insight into channel function. For ion permeation, we exploited K+ and Rubidium (Rb+) permeability of the channel and analyzed using the Clampfit program.

Results

The T153I mutant protein size was identical to Kir7.1 WT protein. The specific protein band confirmed the Kir7.1 amino acid sequence. Live‐cell imaging indicated that both Kir7.1 and T153I are trafficked to the membrane. The IV plot for the Kir7.1 WT showed inward current measured at −150 mV with a mean amplitude of −943.49 ± 180.56 pA (n=6), but the T153I shows negligible inward current of −81.21 ± 20.29 pA (n=9, P=0.0051). Kir7.1 WT zero‐current potential was −62.21 ± 1.66 mV (n=6), however, the average T153I zero‐current potential was −5.98 ± 1.62 mV (n=9, P=6.2x10−12). Rb+ ion, known to permeate readily through Kir7.1 channel, showed current fold‐change of 4.95 ± 0.78 (n=6) in WT, while T153I showed current fold‐change of 2.60 ± 0.50 (n=9, P=0.033). P‐value <0.05 is regarded as significant.

Conclusion

These data indicate that the T153I mutant channel is translated and trafficked to the membrane. However, our data indicate that T153I did not maintain WT zero‐current potential and decreased inward current compared to Kir7.1 WT. Surprisingly, T153I showed Rb+ permeability, albeit less compared to the WT channel. Since T153I is within the inner pore‐lining of the channel, it appears to constrict the channel to make it non‐permeable to K+, but Rb+ can pass through, resulting in a dysfunctional channel.

Support or Funding Information

University of Wisconsin‐Madison Endocrinology and Reproductive Physiology NICHD T32, NIH EY024995, NIH P30EY016665, NIH S10OD018221
Evaluation of In Vitro Models for Pyrrolizidine Alkaloid‐Induced Hepatocellular Injury
Authors: Ramos Kaydee Anne Garrote , Taylor Jonathan , Stamper Brendan D.
Published: April 2020

Objective

Pyrrolizidine alkaloids (PAs) are toxic chemicals found in many herbal medicines, dietary supplements and even food sources such as honey, bread, and tea. Cases of drug‐induced liver injury due to herbal and dietary supplements are relatively common in Asia and are increasing in the United States. The literature suggests that PAs containing macrocyclic structure are more toxic than those with smaller sidechains. The goal of this project was to evaluate two PAs with macrocyclic structure (monocrotaline and retrorsine) and one PA will a smaller sidechain (retronecine) for their potential to elicit toxicity across two in vitro model systems.

Methods

PA toxicity was measured by determining the concentration at which 50% of the cell population was killed (LC50). PAs were dissolved in DMSO and PA‐induced toxicity was measured after 48 hours in two in vitro models: TAMH (transforming growth factor‐α transgenic mouse hepatocytes) and THLE‐2 (transformed human liver epithelial‐2 cells). Statistical analysis of cell viability data was performed using SPSS (Probit regression) to obtain LC50 values.

Results

A decrease in cell viability was observed as PA concentration increased. Interestingly, both cell lines were relatively resistant to PA‐induced toxicity. In many cases only predicted LC50 values for each compound could be generated and are indicated by an asterisk (7.0mM*, 11mM*, and 30.mM* in TAMH for monocrotaline, retrorsine and retronecine, respectively; and 2.2mM, 3.2mM, and >100mM* in THLE‐2 for monocrotaline, retrorsine and retronecine, respectively. Based on the toxicologic parameters, monocrotaline and retrorsine were more toxic (lower LC50 values) than retronecine.

Conclusions

While PAs have been associated with hepatotoxicity, limited toxicity was seen in both the TAMH and THLE‐2 cell lines. Predicted LC50 values were higher than expected, suggesting TAMH and THLE‐2 cells are not good models for studying PA‐induced liver toxicity. Retronecine, which contains a smaller sidechain, was found to be less toxic than the larger compounds. Future work is currently underway to identify biomarkers responsible for the observed resistance to PA‐induced toxicity as potential targets for preventing hepatocellular injury.

Support or Funding Information

This work was supported by the Pacific University Research Incentive Grant Program and an ASPET Summer Undergraduate Research Fellowship (SURF) award at Pacific University.
Examining the mechanism of the methionine E. coli ABC transporter MetNI
Authors: Foronda Matthew
Published: April 2020

To maintain chemical homeostasis, cells need to transport substrates against their concentration gradients through the cellular membrane. In all life forms, ATP‐binding‐cassette (ABC) transporters utilize the energy of ATP binding and hydrolysis to drive this crucial process. When these transporters malfunction, diseases such as cystic fibrosis, Stargardt’s disease (vision loss), and drug‐resistant cancers can develop. Currently, the structure of a number of ABC transporters in different states has been solved, providing snapshots of the transporter in action. Functional analyses oftentimes reinforce structural studies, however in some cases the proposed models are in disagreement. Recent studies suggest that individual transporters may have multiple mechanisms for transport and that the mechanisms between transporters vary significantly.

MetNI is an ABC transporter that imports methionine in bacterial cells. MetI is the transmembrane domain that passes through the cell’s lipid bilayer, and MetN is the site of ATP binding and hydrolysis. Assisting substrate arrival to MetNI is substrate‐binding protein MetQ. Our research focuses on determining the conditions necessary for the interaction of MetNI and MetQ to occur. Using fluorescence anisotropy and fluorescently labeled MetQ, we measure the change in anisotropy as the concentration of MetNI increases. From the anisotropy data we determine the binding dissociation constant of MetNI‐MetQ in the presence and absence of ATP, ADP, and methionine.
Dysregulation of Inositol Synthesis Affects Obesity and Causes Developmental Birth Defects
Authors: Rivera Maria J. , Nguyen LongThy , Eldon Elizabeth D. , Klig Lisa S.
Published: April 2020

Myo‐inositol is a six‐carbon sugar alcohol that is a precursor of phosphatidylinositol (PI), a cell membrane phospholipid. Inositol is synthesized from glucose‐6‐phosphate by the product of the Drosophila melanogaster Inos gene, myo‐inositol‐3‐phosphate synthase (MIPSp). Abnormalities in myo‐inositol metabolism have been implicated in diseases and complications such as type 2 diabetes, polycystic ovary syndrome, and bipolar disorder. Obesity can be induced by a high sucrose diet in D. melanogaster third instar larvae. The hypothesis of this study was that obesity induced in D. melanogaster by high sucrose diet can be alleviated by inositol, either dietary or endogenously synthesized. A float buoyancy assay was used to assess obesity of larvae of three strains: a wild type strain (CS), a homozygous P‐element insertion strain with UASGAL4 5’ of the Inos gene (inosP‐UAS), and a heterozygous inosP‐UAS strain with GAL4 production driven by an actin promoter (inosP‐UAS ; Act5C‐Gal4). This last strain has up‐regulated inositol synthesis. Larval Inos transcript and MIPS protein expression levels in the three strains were confirmed by RT‐qPCR and Western blots. The inosP‐UAS larvae with GAL4 production driven by the actin promoter (inosP‐UAS ; Act5C‐Gal4) have the highest levels of Inos transcript and MIPS protein relative to the homozygous inosP‐UAS insertion larvae which have an intermediate level of expression, and the wild‐type CS with the lowest level of expression. In the float‐buoyancy assay, larvae with increased buoyancy are scored as ‘obese’. Provision of dietary inositol reduced the percentage of ‘obese’ wild‐type larvae from 74.75±1.94 to 63.95 ±1.70. Moreover, up‐regulation of inositol synthesis via inosP‐UAS ; Act5C‐Gal4 dramatically reduced the percentage of ‘obese’ larvae to 13.28±4.42. During these studies, it became apparent that dysregulation of inositol synthesis resulted in developmental abnormalities and birth defects. Attempts to grow inosP‐UAS ; Act5C‐Gal4 larvae to maturity (adult flies) resulted in a surprising outcome – nearly all flies died as pharate adults. Some structural alterations of wings, halteres, and legs were immediately apparent. The few flies that did eclose died within a few days. Scanning electron and light microscope imaging revealed that they all had malformed or missing proboscises. These data give us a better understanding of the importance of the regulation of inositol in obesity and development.

Support or Funding Information

This project is supported in part by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R25GM071638 and by the Frank Schatzlein Endowed Student Award.
Tumor Necrosis Factor‐α Activates Integrin Signaling in Vascular Smooth Muscle Cells via α5 Receptor
Authors: Choi Hyehun , Lamb Fred S.
Published: April 2020

Tumor necrosis factor‐α (TNFα) signaling requires superoxide (O2•−) to be produced extracellularly by NADPH oxidase 1 (Nox1) in vascular smooth muscle cells (VSMCs). Extracellular superoxide dismutase (ecSOD) converts O2•− to hydrogen peroxide (H2O2). It binds to the cell surface by association with either heparin‐sulfate or fibulin‐5, an extracellular matrix protein that interacts with integrin receptors. We previously found that ecSOD knockdown (siRNA) impaired TNFα signal transduction as reflected by MAPK and NF‐κB activation. Targeting of ecSOD also inhibited TNFα‐dependent activation of focal adhesion kinase (FAK) which reflects integrin receptor activation. Here we investigated the mechanism by which TNFα activates integrin signaling in VSMC. We analyzed NF‐κB activation in the presence of selective blocking antibodies (Ab) for integrin receptor subtypes previously shown to be expressed in VSMCs including α4, α5, αv, β1, and β3. TNFα‐induced NF‐κB activation was not affected by blockade of integrin receptors α4, αv, β1, and β3. However blocking of α5 reduced TNFα‐induced NF‐κB activation (control IgG + TNFα 7.36 ± 0.7, anti‐α5 Ab + TNFα 4.78 ± 0.3 fold change, p < 0.05, n = 5). We next sought to determine if α5 integrin is associated with upstream steps of integrin signaling in VSMCs. FAK and integrin‐linked kinase (ILK) are downstream effectors of integrin receptor activation. After 10 min, TNFα induced phosphorylation of both FAK and Protein Kinase B (Akt) which was used as a surrogate for ILK activation. Blockade of α5 receptors decreased TNFα‐induced phosphorylation of both FAK and Akt. Thus, short term exposure of VSMCs to TNFα transactivates α5 integrins and subsequently FAK and ILK‐Akt. We speculate that ecSOD localizes TNFα‐induced H2O2 production to the vicinity of α5 integrins via physical association with fibulin‐5 and this redox environment affects activation of the receptor.