bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation and Characterization of the Pseudomonas aeruginosa Bacteriophage Vanarele
Authors: Rasmussen Reid J. , LaFerriere Holly J. 
Published: April 2020

Antibiotic resistant bacteria have become a major problem in the United States, making treatment of patients a nightmare for hospitals. Phage therapy has reemerged as a possible solution to this ever increasing problem. A novel bacteriophage that infects a biosafety level 2 bacterium, Pseudomonas aeruginosa 15692, was isolated from wastewater in Bemidji, MN and named Vanarele. Antibiotic resistant strains of this bacterium are a major cause of disease in immunocompromised patients. Infection from this bacterium is commonly found in patients with cystic fibrosis and burn wound victims. This bacterium commonly forms biofilms that can be problematic in patient airways and catheters. Characterization of the bacteriophage Vanarele that infects strains of Pseudomonas aeruginosa could provide insight and tools that may be utilized in the treatment of antibiotic resistant infections. The phage was isolated utilizing an enrichment procedure and six rounds of plaque purification. Plaque morphology was examined for the isolated phage and was found to have a slight turbid, halo appearance with an average diameter of 3 mm. Once the bacteriophage had been completely isolated a high titer lysate was harvested. Transmission electron microscopy was then performed to visualize phage size. A capsid size of 40 nm was observed. Currently, the effectiveness of the bacteriophage to break down biofilms formed by multiple strains of Pseudomonas aeruginosa is being tested. The characterization of this phage will add to our knowledge of bacteriophages and may aid in development of new treatments for antibiotic resistant infections.Support or Funding InformationThis work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science.
Determining protein structure with tyrosine bioconjugation
Authors: Moinpour Mahta , Schwartz Jacob 
Published: April 2020

A hallmark of many neurodegenerative diseases like Alzheimer’s disease (AD), Amyotrophic Lateral Sclerosis (ALS) and Frontal Temporal Lobar Dementia (FTLD) is the formation of misfolded proteins into aggregates in the neuronal cells. These proteins often have intrinsically disordered regions which are enriched in a low complexity of amino acids such as Serine, Glycine, Glutamic acid and Tyrosine. Disordered proteins are challenging to study with common techniques such as x‐ray crystallography and NMR. Furthermore, common biorthogonal chemical reactions which mostly target lysine and cysteine residues cannot be used to determine the protein fold due to the low variety of amino acid composition in these proteins. However, a unique characteristic of several disordered proteins involved in neurodegenerative disease is their enrichment for tyrosines. Here we have developed an approach to assess tyrosine accessibility as a measure of levels in native vs misfolded forms of the proteins.To measure tyrosine accessibility we employed the ability of 4‐Phenyl‐1,2,4‐triazoline‐3,5‐dione (PTAD) to conjugate to tyrosine side‐chains. We assessed the targeted labeling of tyrosine’s for peptides, proteins, and cells. We have used this chemical approach to observe the transition of Bovine Serum Albumin (BSA), a well‐characterized and folded protein, to an unfolded state. We found that we could assess the local changes to protein structure by using LC‐MS/MS to quantify levels at each tyrosine position. We could confirm changes using fluorescent detection through click chemistry. Our long‐term goal is combining this approach and proteomics to quantify changes in cells to protein folding in comparable cellular states, such as disease and non‐diseased tissues.Support or Funding InformationNational Institute of Health, American Cancer Society
Microvascular Responses Following 5‐Fluoruracil Chemotherapy Administration
Authors: Hammond Stephen T. , Parr Shannon K. , Hilgenfeld Emma G. , Turpin Vanessa-Rose G. , Ade Carl J. 
Published: April 2020

BACKGROUNDThe pyrimidine antimetabolite 5‐Fluorouracil (5‐FU) is implicated with the second highest rate of cardiotoxicity among cancer chemotherapies, manifesting as vascular vasospasms. Despite this, the pathophysiological mechanisms in which 5‐FU‐induced vasospasms occur is not well understood, but alterations in microvascular endothelial‐dependent and ‐independent pathways have been implicated. This is critical given that decrements in microvascular function are known to occur early in the development of overt cardiovascular disease and like measurements have previously been used to asses pathophysiological mechanisms in numerous patient populations. However, to date, no study has investigated the microvascular function in cancer patients actively receiving 5‐FU chemotherapy. Therefore, we hypothesized that cancer patients treated with 5‐FU would exhibit lower measurements of cutaneous blood flow (CBF) and cutaneous vascular conductance (CVC) following iontophoresis of ACh to that of healthy non‐treated controls.METHODS9 5‐FU treated cancer patients (7 M, 2 F; age 62.8 ± 11.1 yr) and 9 healthy controls (6 M, 3 F; age 56.62 ± 9.9 yr) were recruited for this study. Red blood cell flux was measured non‐invasively in the right forearm via Laser Doppler Flowmetry (LDF) and used as an index of CBF. Endothelial‐dependent vasodilation was elicited via iontophoresis of acetylcholine (ACh). Baseline to peak changes CBF and CVC, derived from CBF and mean arterial pressure were calculated.RESULTSNo significant differences for CBF [5‐FU: 792.78 ± 476.3%, control: 973 ± 682.77%, p = .26] or CVC [5‐FU: 796.76 ± 472.99 %, control: 918.33 ± 638.01.3%, p = .32] were found between groups.CONCLUSIONTo date, our data suggests 5‐FU does not alter Ach‐mediated endothelial‐dependent vasodilation within the cutaneous microcirculation. These findings suggest that endothelial‐independent mechanisms may be linked to 5‐FU cardiotoxicity. Future directions will include a focus on smooth muscle function and vasoconstrictor responses of patients undergoing 5‐FU chemotherapy.
PKA Inhibition Mimics Dopamine 2 receptor‐driven Inhibition of VEGF‐induced Proliferation of Human Retinal Microvascular Endothelial Cells
Authors: Fortepiani Lourdes A. , Adams Kaelyn , Varela Crystal , Akimov Nikolay P. , Sohn Jeong-Hyeon , Renteria Rene C. 
Published: April 2020

Diabetic retinopathy (DR), a leading cause of blindness, results in part from chronically increased expression of vascular endothelial growth factor (VEGF) in the eye in the diabetic state. VEGF increases proliferation of endothelial cells, a critical step in abnormal angiogenesis that contributes to the pathophysiology of DR. We have previously found that activation of dopamine receptors, specifically using a D2‐type receptor (D2R) agonist, inhibits VEGF signaling on human retinal microvascular endothelial cells (HRMECs) and that HRMECs express the dopamine receptor genes DRD1, 2, 3, and 5, but not DRD4. D2R activation normally inhibits the activity of PKA via activation of Gi‐alpha subunits. Here, we determined whether inhibition of PKA, the canonical downstream target of D2R‐agonist activation, suppresses VEGF‐induced proliferation of HRMECs.HRMECs, obtained from CSC, Inc. (Kirkland, WA), were plated in CSC 10% serum complete media in 96‐well plates. After overnight starvation in 0.5% serum media, wells were pretreated with either the D2R agonist quinpirole (Q: 50 nM), the PKA inhibitor 14–22 Amide (PKI: 1 μM), or only 0.5% serum media (C). This and all consecutive steps were performed in 0.5% serum media. After 15 min, VEGF (V: 25 ng/mL) was added to some wells pre‐treated with Q or PKI, resulting in the following groups: C, V, V+PKI, V+Q, Q, and PKI. Proliferation as a percent of control was determined using a WST‐1 assay after 48 hours. As anticipated, VEGF induced proliferation of HRMECs compared to control (1.21 ± 0.13 vs 1, normalized values). This proliferation was blocked by either Q or PKI treatment (0.96 ± 0.12 and 0.94 ± 0.12 vs 1.21 ± 0.13 respectively, p<0.05). Treatment with Q or PKI without VEGF had no effect on proliferation compared to control (1.10 ± 0.13 and 0.97 ± 0.18 respectively).In conclusion, application of the D2R agonist quinpirole or of the PKA inhibitor 14–22 Amide, suppressed VEGF‐induced HRMEC proliferation to control levels. These results suggest that targeting not only specific dopamine receptor activation but also cAMP and PKA signaling systems in HRMECs may be a useful adjunct treatment for blocking VEGF‐induced HRMEC activation in DR.Support or Funding InformationNIH R01 EY023290 and UIWRSO Faculty Development Grant
Phytoestrogen Coumestrol as an Anticancer Therapy Against Triple‐Negative Inflammatory Breast Cancer
Authors: Rodriguez-Martir Keishla M. , Rivera Colon Jose E. , Colon Ortiz Ayeisha N. , Peterson Esther A. 
Published: April 2020

Breast cancer is the second leading cause of death in females in the United States, being Inflammatory Breast Cancer (IBC) the most aggressive subtype. The 5‐year survival rate is 40% for IBC as compared to 85% survival rate in patients with other subtypes. The poor prognosis for patients with IBC emphasizes the need to better understand the molecular signature of this disease with the goal of developing effective targeted therapeutics. To this date, there is no effective targeted therapeutics, especially for those patients that account for approximately 20–40% of IBC. Those cases, triple negative IBC (TN‐IBC), express an alternative isoform of estrogen receptor alpha, estrogen receptor alpha‐36, and GPR30. Importantly, several studies have shown that estrogen can activate non‐genomic signaling pathways involved in the acquisition of oncogenic phenotypes such as increased motility and invasion in triple negative (TNBC) and TN‐IBC. In fact, recent studies have shown that phytoestrogen like Cou, structurally similar to estradiol (E2), induces cell cycle arrest and apoptosis in TNBC. We hypothesize (1) that estrogen non‐genomic signaling has an active role in the aggressive metastatic phenotype of IBC, and (2) that Cou has anticancer activity by inhibiting estrogen non‐genomic signaling in IBC. To test this hypothesis, TN‐IBC cells were treated with E2 or Cou and the effect on phosphorylation of downstream kinases (ERK and AKT) was analyzed by Western blot. Also, functional assays had been performed to determine the effect of treatment with E2 alone, Cou alone, and in combination on migration, proliferation and tumor emboli formation. Immunofluorescence in TN‐IBC and Western Blot of breast cancer cell lines showed expression of estrogen receptors. In addition, preliminary observations demonstrated that upon E2 treatment there was an activation of ERK and AKT kinase in both TN‐IBC and TNBC cell lines. Finally, treatment using previously reported IC50 (50μM) of Cou caused a decrease in cell viability in breast cancer cell lines. Also, in comparison to E2, Cou decrease migration, proliferation, and tumor emboli formation in TN‐IBC. In summary, TN‐IBC cells are responsive to E2 treatment making this signaling pathway attractive for the development of innovative therapeutic strategies. Importantly, these Cou preliminary studies show a potential anticancer activity in TN‐IBC cell lines.Support or Funding InformationUPR Fondos Institucionales para la Investigación (FIPI) and RISE program (5R25GM061151‐18)
Evidence for vascular microbleeds in brains following repeated mild traumatic brain injury
Authors: Nahirney Patrick Charles , Trivino Juan , Christie Brian R. 
Published: April 2020

Mild traumatic brain injury (mTBI) is produced by the rapid movement of the brain within the skull. It is believed that each mTBI leaves the brain in a vulnerable state to subsequent injuries (repeated‐mTBI; r‐mTBI), and can precipitate more serious neurological conditions. However, a mechanism responsible for this vulnerable state is currently lacking, and the mTBI process itself remains poorly understood. The current experiments used an Awake Closed Head Injury (ACHI) model we have recently developed to study r‐mTBI. For each ACHI procedure (n = 8 per animal) a neurological assessment was performed to assess basic levels of consciousness, balance, and sensory perception. Our data demonstrate that administering the ACHI protocol 8 times produces robust, but non‐cumulative neurological deficits. One day following mTBI, rats were perfusion fixed, vibratome sliced (50 μm), and processed for immunohistochemistry. Confocal and light microscopic imaging revealed that r‐mTBI is accompanied by diffuse microbleeds in cortical and subcortical regions that ranged in size from 20 to 100 μm. These microbleeds were characterized by the presence of red blood cells and the blood protein, fibrinogen, in the surrounding perivascular space. The parenchyma around these microbleeds was positive for reactive and phagocytic microglia. In addition, the brains of animals following r‐mTBI were unique in that they also contained ‘rod’ type microglia, similar to those normally observed in Alzeheimer‐like dementias. These observations indicate that repeated concussions produce ruptures in small blood vessels in the brain and these likely contribute to both immediate and long‐term cognitive deficits.Support or Funding InformationCIHR, NSERC
Habitual Exercise and ETB Receptor Responses in Postmenopausal Women
Authors: Welti Laura M. , McGinty Shane J. , Kuczmarski Andrew V. , Nathaniel Sangeetha , Shoemaker Leena N. , Wenner Megan M. 
Published: April 2020

Age‐related declines in endothelial function can be reversed with habitual exercise (HEx) in older men by attenuating the vasoconstricting effects of endothelin‐1 (ET‐1), attributable in part by enhanced ETA receptor activity. Our laboratory has recently demonstrated that ET‐1 contributes to age‐related endothelial dysfunction in postmenopausal women (PMW) via the ETB receptor (ETBR). Although there is evidence for sex‐specific differences in ET‐1 receptor activity with aging, the impact of habitual exercise (HEx) on ET‐1 receptor responses in PMW has yet to be established.PURPOSETo test the hypothesis that ETBR responses would be attenuated in HEx PMW.METHODSIn a retrospective analysis, 30 PMW were grouped by self‐reported weekly exercise. We measured vasodilatory responses to local heating of the skin (laser Doppler flowmetry) during microdialysis perfusions of lactated Ringer’s (Control) and ETBR blockade (BQ‐788, 300nM) in 15 low (LEx: 2±2 d/wk, 57±4 yrs) and 15 high (HEx: 6±1 d/wk, 60±5 yrs) frequency exercisers. Cutaneous vascular conductance (CVC) was calculated during the plateau phase of local heating (42°C), and normalized to maximal vasodilation achieved by perfusion of sodium nitroprusside (28mM) and heating to 43°C. We analyzed the impact of weekly exercise frequency on vasodilatory responses to ETBR blockade using a two‐way ANOVA. Data presented are mean ± standard deviation.RESULTSGroups were well‐matched for body mass index (BMI) (LEx: 25±4 vs HEx: 24±2 kg/m2, P=0.31), resting heart rate (LEx: 60±8 vs HEx: 54±9 bpm, P=0.14), and resting mean arterial pressure (LEx: 90±10 vs HEx: 90±8 mmHg, P=0.96). Microvascular endothelial function was similar between LEx (control: 87±9 %CVCmax) and HEx (control: 88±6 %CVCmax, P=0.86). ETBR blockade improved vasodilation in LEx (BQ‐788: 93±7 %CVCmax, P=0.01 vs. control) but not in HEx (BQ‐788: 91±7 %CVCmax, P=0.11). To account for differences in age and time since menopause, we performed a sub‐analysis of age and BMI‐matched early PMW (<5 years since menopause) PMW (LEx, n=4: 53±2 yrs, 24±4 kg/m2; HEx, n=5: 53±3 yrs, 23±2 kg/m2). This sub‐analysis revealed a similar trend of enhanced vasodilation during ETBR blockade in LEx (control: 88±3 vs BQ‐788: 96±4 %CVCmax) but not HEx PMW (control: 87±6 vs BQ‐788: 85±13 %CVCmax; drug*exercise, P=0.07).CONCLUSIONBlocking ETBR improved microvascular endothelial function in LEx, indicating a removal of the vasoconstricting effects of ETBR in LEx but not HEx PMW. Therefore, these data suggest that exercise may attenuate ETBR‐mediated vasoconstriction in PMW. Additional data are needed to determine the impact of exercise on ETA receptor responses in PMW.Support or Funding InformationSupported by NIH R01 HL 146558, P20 GM 113125, AHA 16SDG30700015, and University of Delaware Research Foundation
Changes in Splanchnic Hemodynamics Contribute to the Cardiovascular Responses Following Activation of Bradykinin‐Sensitive Pericardial Afferents
Authors: Martin Doug , Roessler Kyle 
Published: April 2020

Sympathetic drive is a major controller of cardiovascular function. The heart performs an important sensory function via a variety of cardiac receptors that modulate cardiovascular responses by both neural and humoral reflexes. Bradykinin‐sensitive sympathetic afferent nerves from the heart can trigger marked increases in efferent sympathetic nerve activity to some cardiovascular target organs such as the kidney, arteries and veins. We reported previously that pericardial injection of bradykinin increased venous tone and cardiac output. The splanchnic vascular compartment plays an important role in control of venous return and cardiac output. This work tested the hypothesis that activation of bradykinin‐sensitive pericardial afferents modulates splanchnic hemodynamics.Sprague Dawley rats were anesthetized with a cocktail of alpha chloralose (80 mg/kg) and urethane (800 mg/kg). Catheters were placed in the femoral artery and vein to record arterial pressure (MAP) and heart rate (HR). A PE10 catheter was placed in the pericardial space for the injection of bradykinin (BK), an agent known to stimulate cardiac sympathetic afferents. The chest was closed and the rats allowed to breathe spontaneously. The portal vein and mesenteric artery were exposed via a midline laparotomy and fitted with transit time flow probes. After stabilization, the blood pressure, heart rate, portal venous flow (PF) and mesenteric flow (MF) responses to pericardial injection of BK or vehicle were recorded.Pericardial injection of saline vehicle had little effect (MAP: 2±2 mm Hg; HR: 13±6 bpm; PF: −5±1%; MF: −3±1%). In contrast, pericardial injection of BK (10 ug/kg) increased MAP (16±3 mm Hg) and HR (28±5 bpm). After an initial transient decrease, PF (10±2%) and MF (5±2%) were increased during the plateau phase of the MAP response (60–120 seconds).We interpret these data to indicate that activation of bradykinin‐sensitive pericardial afferents increases portal venous flow. This finding is consistent with the view that activation of the cardiac sympathetic afferent reflex elicits a venoconstrictor response to increase venous return from the splanchnic compartment.Support or Funding InformationSupported by NIH R01 HL136741‐03, and the Basic Biomedical Sciences program of the Sanford School of Medicine.
Making Functional Predictions Using Local Spatial Arrangements in the Haloacid Dehalogenase Superfamily
Authors: Ruffner Lydia Armentha , Pina Manuel , Beuning Penny J. , Ondrechen Mary Jo 
Published: April 2020

Beginning with the sequencing of the human genome in the 1990s, major efforts have been underway to expand significantly upon our current working knowledge of proteins. Between the years 2000–2015, the Protein Structure Initiative (PSI) solved 15,000+ protein structures, but most of them have unknown or uncertain biochemical function or have an incorrect assigned putative function. With all these structures, methodology must be developed to functionally annotate these proteins in order to identify potential applications, such as bioremediation and understanding cellular processes. Enzymes in the Haloacid Dehalogenase Superfamily (HADSF) possess a wide range of functions, including phosphatases important in cell membrane biosynthesis and dehalogenases that possesses the ability to detoxify and degrade halogenated compounds. The Ondrechen Research Group (ORG) at Northeastern University has successfully developed methodology that is being used to predict biochemical function for Structural Genomics (SG) proteins in the HADSF. These methods are Partial Order Optimum Likelihood (POOL) and Structurally Aligned Local Sites of Activity (SALSA). POOL is a machine learning method that uses the electrostatic features and metrics from THEoreticalMicroscope Anomalous TItration Curve Shapes (THEMATICS), ligand binding pocket and geometric features from ConCavity, and the evolutionary scores and phylogenetic trees from INformation‐theoretic TREe traversal for Protein functional site IDentification (INTREPID) to make the predictions about which residues are catalytically active or otherwise important for protein function. SALSA uses the functional residue predictions for SG proteins obtained from POOL and aligns them with consensus signatures from known enzyme subfamilies according to the local spatial arrangement of predicted residues at the active site. So far for the HAD superfamily, using SALSA we have made predictions for 20 SG proteins: one dehalogenase, eight sugar phosphatases, three NagD‐like phosphatases, four P‐Type ATPases, and four soluble epoxide hydrolases. These predictions will be experimentally validated by biochemical assays to establish the function of each protein and to verify our computational approach to protein function prediction. The ability to predict computationally the biochemical function of protein structures of unknown or uncertain function adds tremendous value to genomics data.Support or Funding InformationThis project is funded by NSF CHE‐1305655, CHE‐1905214, and CHE‐1757078.