bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dissecting Amino Acid Osmolytes: Is a Zwitterion Required for Increasing Protein Thermal Stability?
Authors: Canepa Jacob , Wilkinson Steven 
Published: April 2020

Amino acids are an important class of osmolytes that demonstrate the ability to stabilize certain proteins under acute thermal stress. However, little consensus exists on what structural aspects produce this phenomenon. It has been proposed that the stabilizing nature of amino acids emerges from their zwitterionic nature at neutral pH, but little evidence has supported this view, especially in regards to the breadth of its applicability to proteins of varying structure and composition. In this report, we structurally dissect the amino acids L‐alanine and β‐alanine to determine functional group contributions to protein‐stabilizing activity on two structurally dissimilar proteins: human C‐reactive protein (CRP) and human myoglobin. The stabilizing effects of the osmolytes (and their structural derivatives) were quantified by Differential Scanning Fluorimetry (DSF) in three metrics: protein melting temperature (Tm), standard free energy change (ΔGo), and “m”‐values. Our results indicate that the zwitterionic theory holds for myoglobin, as removal of either the carboxylate or amino moiety from the amino acid abrogated all thermal stabilizing effects. Furthermore, substitution of the carboxylate group with a hydroxyl group did not restore protein‐stabilizing activity, arguing for the zwitterionic requirement for amino acid‐mediated thermal stabilization. However, the results with CRP were significantly different from those observed with myoglobin. In the case of CRP, the amino acid carboxylate proved to be the dominant stabilizing group, as compounds lacking the amino group conferred greater thermal stability than the complete amino acids. These results affirm that the zwitterionic theory is limited in its ability to describe amino acid‐mediated effects on protein thermal stability and suggests that the nature of the interactions between amino acid osmolytes and proteins is highly dependent on protein structure. These results may have significant implications for the development of chemistries to stabilize diverse protein targets in biospecimens for diagnostic applications.Support or Funding InformationCalifornia State University Program for Education and Research in Biotechnology (CSUPERB)Chevron Biotechnology Applied Research EndowmentBill and Linda Frost Fund
Mature Adipocytes and FGF2 Influence Angiogenic Processes
Authors: Lowke Makenzie Taylor , Cortez Xiomara , Garcia Michelle Renee 
Published: April 2020

The process of wound healing requires the transportation of substrates needed for the rebuilding of tissue through appropriate vessel formation; angiogenesis. Adipose tissue extract is often used to facilitate the wound healing process, which is attributed to its potent angiogenic properties. Most angiogenic process are regulated through growth factors such as vascular endothelial growth factor (VEGF), angiopoietin, and fibroblast growth factor (FGF). Fibroblast growth factor has been utilized to facilitate wound healing in the biomedical community, which is one of the growth factors synthesized and secreted by adipocytes. Therefore, it was hypothesized that mature adipocytes augment angiogenic processes in dispersed adipose tissue in the presence of FGF2 (basic). Adipose tissue was collected from 4 prepubertal female pigs (gilts) and enzymatically dispersed (collagenase type II). Cells were separated into 2 groups 1) mature lipid‐filled adipocytes (MA) and 2) all other cell types (OC) i.e., vascular endothelial cells (VEC), preadipocytes, and fibroblasts. The OC were incubated for 24 hr in cell culture media with 10% fetal bovine serum (FBS) to promote attachment and angiogenic processes. After the first 24 hr incubation period, media was removed and replaced with 2% FBS and MA were added with or without FGF2 (10−11 M) or rabbit polyclonal anti‐FGF2 (1:100 dilution) and incubated for an additional 24 hr. The MIXED procedure of SAS was utilized to determine the effect of treatment on in vitro angiogenic cellular processes. The addition of MA alone augmented cellular angiogenic processes including elongation, sprouting, formation of nascent tubes, and vessel tube formation (P < 0.05). However, vessel tube formation in the FGF2 treatment group was lower than MA alone (P < 0.05), which was ameliorated, in part, with FGF2 antibody treatment. Mature adipocytes appear to augment angiogenic processes; however, FGF2 may diminish or impair the progression of vessel tube formation.Support or Funding InformationUSDA LEADERS 2 Award #2017‐38422‐27298
Peroxidase activity along soil depth of a metal contaminated brownfield in New Jersey
Authors: Dyson Eshariah N. , Idrovo Cesar , Hagmann Diane , Krumins Jenifer , Goodey Nina 
Published: April 2020

The peroxidase enzyme is a member of the oxidoreductase family of enzymes. It has been referred to as a remediation enzyme, as more peroxidase activity is observed when soils are experiencing oxidative stress. At Liberty State Park, a New Jersey brownfield, there is a section of the park that is closed to the public because of high levels of metal contamination. Despite the presence of metals in the soil, much of the area supports plant life. The goal of this study is to determine how the peroxidase activity and metals are distributed along soil depth and to compare results from a forested and barren area of the site. To do this, we collected three 10 cm deep soil cores from each of the two sites. Using the cores, we examine the peroxidase activity and metal concentrations of each 2 cm cross section. We found a similar trend in distribution along depth for metal concentration and peroxidase activity in the forested site. The barren site had a measurable peroxidase activity with a high metal concentration found in the top 2 cm cross section. These studies can serve as tools to understand the relationship between peroxidase activity, metal load, and soil depth and determine how these factors effect plant growth.Support or Funding InformationNSF Grant CBET #1603741
Phosphorylation of Serine 263 in the Human Choline Transporter 1 alters the transporter’s function
Authors: Rizvi Midhat Ali , Arutyunov Denis , Caluseriu Oana , Leslie Elaine , Cordat Emmanuelle 
Published: April 2020

Disruptions of the neuromuscular junction result in congenital myasthenic syndromes (CMS). CMS is characterized by akinesia, apneas or even death. CMS type 20 is caused by mutations in the SLC5A7 gene which encodes for the choline transporter (CHT1). The 13 transmembrane domain CHT1 protein is thought to be phosphorylated at several sites but the role of phosphorylation remains unknown. We have recently reported the missense recessive substitution c.C788>T in SLC5A7 in a family with a history of CMS using whole exome sequencing. This mutation encodes a p.S263F substitution in CHT1 and results in a complete loss of transport function despite a normal plasma membrane localization. To assess whether 1) the p.S263F substitution alters CHT1 phosphorylation state, and 2) a change in phosphorylation results in altered transport function, three substitutions were introduced into CHT1: p.S263A, and phosphomimetic p.S263D and p.S263E. Upon expression in HEK293 cells, we found that p.S263A mutant retained a high level of transport activity while the p.S263D and p.S263E mutants were non‐functional, suggesting that S263 phosphorylation inhibits choline transport. Immobilized metal affinity electrophoresis supports that wild‐type CHT1 may be more phosphorylated than p.S263F CHT1 mutant. These results support the essential role of Serine 263 residue in CHT1 choline transport possibly via altering its phosphorylation status.Support or Funding InformationWomen and Children’s Health Research Institute, Stollery Children’s Hospital Foundation, Canadian Institute of Health Research & The University of Alberta
IMPLICATING RIBONUCLEASES IN MAMALIAN TAILED MIRTRON BIOGENESIS
Authors: Zia Farid , Flynt Alex 
Published: April 2020

MicroRNAs are pervasive small noncoding ~22 nt gene regulators that collectively control gene expression by destabilizing mRNA through repressing translation. Since they are broadly involved in cell differentiation and maintenance, they are linked to most diseases including the major causes of mortality, heart disease and cancer. Several thousand miRNA genes have been reported in mammals that typically generated thorough a canonical pathway involving sequential cleavage by Drosha and Dicer, however, there are many non‐canonical miRNAs that mature through alternative pathways. Mirtrons are the most abundant class of atypical miRNA that bypass Drosha cleavage step and instead are produced by splicing. Following lariat debranching, the host intron RNA can be exported to cytoplasm for Dicer processing. There are 3 classes of mirtrons: conventional, 5′‐tailed, and 3′‐tailed. In tailed mirtrons, splicing defines only pre‐miRNA hairpin end. While a mechanism has been reported for 3′‐tailed mirtrons, processing of 5′‐tailed varieties remains a mystery. Here, we studied the biogenesis of human and mouse 5′‐tailed mirtrons to implicate RNases in tail processing. Through a mutational approach we have identified endocnucleolytic cleavage of precursors to be important to processing of 5′‐tailed mirtrons. This is a novel mechanism that contrasts with what has been shown for 3′ tailed mirtrons. Our findings shed light on the most substantial variety of mirtrons encoded by the human genome.Support or Funding InformationThis work was supported by the National Institute of Health and also Mississippi INBRE program (R15GM120716‐01).
Tissue Factor Pathway Inhibitor (TFPI) release by bovine, ovine and porcine unfractionated heparins in primates. Comparative studies at mass and potency adjusted intravenous dosing
Authors: Kouta Ahmed , Bontekoe Emily , Duff Richard , Jeske Walter , Hoppensteadt Debra , Yao Yiming , Fareed Jawed 
Published: April 2020

BackgroundUnfractionated heparin (UFH) is a highly sulfated glycosaminoglycan (GAG) that consists of repeating disaccharide units, containing iduronic acid (or glucuronic acid) and glucosamine, exhibiting variable degrees of sulfation. UFHs release tissue factor pathway inhibitor (TFPI) which inhibits extrinsic pathway of coagulation by rapidly inactivating of Factor Xa and the Factor VIIa/TF complex. Bovine mucosal heparins (BMH) and ovine mucosal heparin (OMH) are currently being developed for re‐introduction for both medical and surgical indications. In contrast to the porcine mucosal heparins (PMH), the active pharmaceutical ingredient (API) BMH exhibits a somewhat weaker anti‐coagulant activity as cross‐referenced against PMH. In this study, we determined the TFPI antigen release level by various dosages of UFHs using ELISA method. Also, we projected that potency adjusted BMH at equivalent dosage has the same TFPI release profile compared to OMH and PMH.Materials & MethodsBMH, OMH and PMH were obtained from various commercial sources. Potencies of each heparin were determined by an amidolytic anti‐Xa assay in relation to the USP heparin standard. Individual groups of primates (n=4) were administered 0.5 mg/kg or (50 or 100) U/kg dosage of bovine, ovine or porcine UFH via intravenous route. Blood samples were collected at baseline, 15, 30, 60 and 120 minutes post‐administration. Tissue Factor Pathway Inhibitor (TFPI) antigen levels were measured using a commercially available sandwich ELISA methods. All results were compiled as mean ± SD.ResultsAt a gravimetric level (0.5 mg/kg), BMH exhibited a lower TFPI level than OMH & PMH at all time points with a maximum value of (200 ng/ml) at 15 mins post drug administration. OMH and PMH showed comparable TFPI levels at all time points with maximum values of (250 ng/ml) and (265 ng/ml) respectively at 15 mins post drug administration. However, USP cross‐referenced anti‐Xa potency adjusted based dosing showed comparable TFPI antigen levels for all three UFHs at all time points with a maximum value of 250 ng/ml at 15 mins post drug administration. At 60 mins post drug administration, potency adjusted BMH showed slightly higher TFPI level of (200 ng/ml) compared to both OMH & PMH.ConclusionTissue factor pathway inhibitor (TFPI) levels at 15 minutes after IV administration of 0.5 mg/kg heparins to non‐human primates showed that both PMH and OMH release comparable TFPI antigen levels which were significantly higher than BMH. Potency equated dosing resulted in comparable TFPI release profiles for all three heparins. Therefore such dosing may provide uniform levels of anticoagulation for the parenteral indications for UFHs. These observations suggest that potency equated bovine heparin has comparable release of TFPI and therefore is biosimilar to the OMH and PMH.
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 InformationSupport 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 receptorsSupport or Funding InformationFlorey 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 InformationThis 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

IntroductionThe 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.MethodsBlood 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).ResultsExperiments 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.ConclusionsIL‐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 InformationAustralian Research Council (FT170100363)National Health and Medical Research Council (GNT 1079680)Rebecca L Cooper Medical Research Foundation