bluuebunny/crossref_metadata_2025 huggingface dataset:
The Detection of Antibiotic Resistance Genes in Soil Samples from Rural and Urban Environments
Authors: Schuster Noah Avery
Published: April 2020

The soil is an extremely large reservoir for microbial diversity, containing large numbers of resistant microorganisms that possess a variety of antibiotic resistance genes. Determining the composition and distribution of these genes within the soil provides information to further our understandings of antibiotic resistance evolution and development. Profiling the soil resistome can also reveal previously unknown threats to public health. We hypothesize that a larger number of resistance genes would be present in urban environments when compared to rural environments. In this study, soil was collected from twelve locations, with six locations in a rural environment and the other six in an urban environment. Soil gathered from each location was then characterized based entirely on texture and pH, followed by enrichment with tryptic soy broth containing tetracycline and incubated for three days at room temperature. Following DNA extraction from each enrichment, the presence of different tetracycline resistance genes (n = 14) was evaluated. A total of 23 tetracycline resistance genes were found to exist throughout 11 locations and the most frequently detected gene was tet(L) (30%), followed by tet(M) (26%), tetA(P) (22%), tet(O) (9%), tet(B) (9%), and tet(A) (4%). Each sequence from this study had >97% alignment when compared against known sequences of tetracycline resistance genes. In dividing sampling locations into rural and urban environments, only 12 (52%) tetracycline resistance genes were found in rural soils and 11 (48%) tetracycline resistance genes were found in urban soils. Data collected from this study does not seem to indicate any relationship between soil texture or pH versus the amount of genes present in each location. The location having the greatest number of genes (n = 5) is near local septic systems which might explain the large number of genes present. Based on function, 4 efflux and 2 ribosomal protection genes were detected; however, there is no evidence to suggest whether these genes are found more in rural or urban environments. Taken together, our data to‐date suggests that the number of tetracycline resistance genes is equally distributed in the rural and urban environments. This study hopes to continue assessing the soil resistome by analyzing DNA extracts from carbenicillin and chloramphenicol enrichments, in part with a microbial community analysis using next‐generation sequencing.

Support or Funding Information

Asher Fund, DePauw University
Evidence and Inhibitor Studies of Involvement of Src and EGFR in Estrogen Receptor Induced ERK1/2 Activation in Ovarian Cancer Cell Proliferation Stimulated by Cadmium Chloride.
Authors: Steinke Kira , Huff Mary
Published: April 2020

Cadmium Chloride, Cadmium, a naturally occurring heavy metal, is a toxic industrial and environmental pollutant commonly found in both ground and industrial waste water. Studies have shown that cadmium is also a metalloestrogen that can bind to the human estrogen receptor (ER), stimulate DNA synthesis, and increases cellular proliferation in ovarian cancer cell lines. Furthermore, it has been shown that cadmium stimulates ERK1/2 activity in an ER‐dependent manner. To determine if cadmium stimulates proliferation in ovarian cancer cell lines, OVCAR3 and SKOV3 cells were treated with varying concentrations cadmium chloride, and growth was measured by a cell proliferation assay. The results support that cadmium induces cellular proliferation at environmentally relevant concentrations (0.001–1.0 uM) within 24 hours of treatment. It was also observed that treatment with 1 nM of cadmium for 5 minutes activates ERK1/2 phosphorylation. We are now in the process of performing inhibitor studies to determine if Src and EGFR are also involved in ER dependent cadmium‐stimulated cellular proliferation in ovarian cancer cells.

Support or Funding Information

Bellarmine University, Student Government Association
PIP2 Binds Multiple Cationic Sites of ENaC
Authors: Archer Crystal R. , Carver Chase M. , Enslow Benjamin T. , Stockand James D.
Published: April 2020

Phosphatidylinositol 4,5‐bisphosphate (PIP2) regulates the activity and gating of many different ion channels to include the epithelial Na+ channel (ENaC). The negatively charged phosphoryl groups of the PIP2 headgroup are expected to interact with cationic residues of the cytoplasmic tails of ENaC. To date, understanding of the biophysical interaction between ENaC and PIP2 has been limited to indirect functional assays. Consequently, the precise binding sites and their affinities have not been defined. We began this study using secondary structure prediction tools to show that helical conformation that may cluster the cationic residues of ENaC cytoplasmic tails. Such clustering would form an electropositive region attractive to the PIP2 headgroup. We used a CIBN/CRY2‐5‐ptase optogenetic dimerization system in HEK cells to show that the C5 phosphoryl group of PIP2 plays a role in regulating ENaC activity. To more precisely define the PIP2 binding sites of ENaC, we designed short peptides corresponding to the cationic clusters of ENaC. Steady state intrinsic fluorescence ligand binding data showed that PIP2 interacted with the extreme amino terminus of the β‐ENaC subunit (βN1 peptide) and the amino and carboxy termini of γ‐ENaC nearest the transmembrane domains (γN2 and γC peptides, respectively), but no significant changes were observed with the other peptides or mutant controls. Microscale thermophoresis data revealed that the βN1 and γN2 sites has the strongest interaction with the PIP2 headgroup with Kd ~5.2 and 15 μM, respectively, whereas the γC site interacts with much weaker affinity. Again, no interaction was observed between PIP2 and the other peptides and mutant controls. These results are consistent with PIP2 binding ENaC to stabilize the open conformation of channel via moderate and weak electrostatic interactions within the cytoplasmic termini.

Support or Funding Information

This work is supported by NIH/HHLBI T32 HL07446 to CRA and JDS, and NIH/NIDDK R01 DK987460 and DK117909 to JDS.
Effects of alcohol pre‐exposure on cocaine locomotor sensitization
Authors: Joe Tawnjerae R. , Silva Daniel da Silva E , Alvarez Veronica A.
Published: April 2020

Cocaine addiction is a chronic disorder characterized by compulsive use despite negative consequences and effects about 1 million people in the United States. Cocaine use is commonly preceded by experiences with the use of alcohol. In fact, about 90% of patients with cocaine use disorder have used alcohol. To understand the biological basis of this additive drug interaction, we investigated if pre‐exposure to alcohol modulates cocaine locomotor sensitization (CLS), a drug‐contingent learning that shares similar neurocircuitry with cocaine taking and relapse. We first tested the ability of voluntary alcohol consumption to modulate CLS. Mice were given intermittent access to 20% alcohol and water for three weeks followed by the CLS protocol that consisted of 5 daily injections of 20 mg/kg of cocaine. Results showed no difference in CLS between alcohol drinkers and alcohol naive controls. Second, we tested whether mice previously sensitized to alcohol increase their CLS response. We found that mice pre‐sensitized to alcohol showed a greater acute response to cocaine and a faster development of cocaine sensitization, suggesting that changes promoted by these drugs in the brain rely on similar neurocircuitry. These experiments shed light on the underlying mechanism by which prior exposure to alcohol facilitates cocaine use later in life.
Kidney mitochondrial redox state 3 days following uninephrectomy in rats determined by cyro‐fluorescence optical imaging.
Authors: Cowley Allen W. , Yang Chun , Shimada Satoshi , Zheleznova Nadya , Mehrvar Shima , Foomani Farnaz , Mostaghimi Soudeh , Ranji Mahsa
Published: April 2020

Uninephrectomy (UNX) is known to result in structural and metabolic changes to the remaining kidney although it is uncertain if this results in changes in the mitochondrial redox state and how soon such changes may occur. A custom designed fluorescence cryo‐imaging technique was used to quantitatively assess the effect of UNX on the mitochondrial bioenergetics of the remaining kidney. Responses were obtained in kidneys of normal Sprague Dawley (SD) rats, Dahl salt‐ sensitive (SS) rats, and SS rats in which the gene for NADPH‐oxidase isoform 4 (NOX4) was knocked out (SSNOX4−/−). Kidneys were snap frozen and the intrinsic fluorescence of two coenzymes, Nicotinamide Adenine Dinucleotide (NADH) and oxidized Flavin Adenine Dinucleotide (FAD) were optically acquired and 3D images created to characterize the NADH/FAD redox ratios (RR) of each kidney 3 days following UNX. Each of the strains exhibited similar increases of kidney weights averaging 17% after 3 days of UNX. It was especially notable that following UNX, SD and SSNOX4−/− rats exhibited global reductions of the RR ratios (P<0.05) with a similar tendency observed in SS rats (P<0.08) indicative of increased reactive oxygen species (ROS) production. The unexpected reduction of the mitochondrial RR in the remaining kidneys of SSNOX4−/− rats indicates that mechanisms independent of H2O2 produced from NOX4 may be largely response for this global increase of renal oxidative stress. We propose that enhanced mitochondrial bioenergetics due to increased tubular workload of the remaining kidney becomes the dominant source of increased ROS following UNX and could represent an important hypertrophic signaling mechanism.

Support or Funding Information

HL‐116264
Identification of Molecules by Spectral Imaging
Authors: Alshammari Qamar Aqeel , Pala Rajasekhar , Katzir Nir , Nauli Surya M.
Published: April 2020

Spectral imaging (SI) is a powerful technique which primarily uses the wavelength to identify and quantify the molecules precisely with their exact location. It facilitates the identification of chemical substances and studying their properties through analyzing the way they interact with light. The study of light interaction with elements is called spectroscopy; spectroscopy examines how light behaves in the target and recognizes materials based on their spectral signatures. These spectromicroscopic methods provide a quantitative analysis of a biological sample and its substructures with a depth of analysis that no other analytical technique can determine from the same sample. Therefore, we hypothesize that identifying the exact location and quantity of molecules present in the given biological (LLCPK‐1 cells) samples can be done by using a SI system. In this study, we first measured the exact UV‐Vis absorption and fluorescence spectra of our test substances of Rhodamine 6G and Doxorubicin, independently. After that, we used the Quadrupole Time‐of‐Flight Liquid Chromatography/ Mass Spectrometer (Q‐TOF LC/MS ) system to quantify the maximum and minimum detectable concentrations of Rhodamine 6G and Doxorubicin by checking the molecular weight of mass‐spectra. In addition, we used High Performance Liquid Chromatography (HPLC) system to quantify the same chemicals based on their UV spectrum at 218 nm. Finally, we used SI microscope to determine the exact amount and location of the molecules present in the cells. For Rhodamine 6G and doxorubicin, we started with the minimum detectable concentrations by Q‐TOF and consider it as a maximum limit for the SI. Using the SI we were able to detect the specific location of Rhodamine 6G was in the cytoplasm and Doxorubicin in the nucleoplasm. In conclusion, SI was able to detect much more lower concentrations of Rhodamine 6G, in the cytoplasm, and Doxorubicin, in the nucleoplasm, based on the spectrum in comparison to Q‐TOF LC/MS.

Support or Funding Information

Saudi Arabian Cultural Mission
Functional Connectivity of Prelimic Prefrontal Cortex and Rostral Ventromedial Medulla for Descending Pain Modulation
Authors: Smith Angela , Flohrschutz Austin , Riegel Arthur , Largent-Milnes Tally , Vanderah Todd
Published: April 2020

Chronic pain is a health condition that can seriously impact quality of life. To develop better treatments for chronic pain, a better understanding of the mechanisms behind pain perception is needed. Current models of descending pain modulation show the prelimbic prefrontal cortex (plPFC; area 32V) synapsing onto cells within the periaqueductal grey (PAG) which then synapses onto the rostral ventromedial medulla (RVM). The RVM contains ‘on’ and ‘off’ cells which respond to pain stimuli by respectively activating or inhibiting pain perception. We aim to functionally characterize a hypothesized direct projection from the plPFC to the RVM first described in 1997. Anterograde tracing was performed in the plPFC to determine the anatomical connectivity of the two regions. Similarly, we transfected the plPFC with hM4Dq DREADDs fused with mCherry under the control of CaMKIIa promoters to label glutamatergic Layer 5 projection neurons and determine functional connectivity of the purported circuit in animals with a spared nerve injury (SNI). These receptors are inert unless acted on by a designer drug, such as CNO. We used reverse microdialysis to locally deliver CNO into the RVM and activate DREADDs on neurons with direct projections from the plPFC while collecting released neurotransmitter and performing reflexive pain behaviors assays. This strategy allowed for avoidance of systemic/off target activation of plPFC neurons projecting to a different CNS region. Anatomical and microanalysis pilot data indicate a direct connection from plPFC to the RVM.

Support or Funding Information

Funding provided by the University of Arizona Department of Medicinal Pharmacology, University of Arizona Undergraduate Biological Research Program, and the ASPET Summer Undergraduate Research Fellows Program.
Antioxidant Capacity and Antiproliferative Activity of Morchella esculenta in Human Ovarian Adenocarcinoma Cells
Authors: Scales Karissa , Schlacks Alexander , Parr Tyler , Soendergaard Mette
Published: April 2020

Morchella esculenta is a coveted wild edible mushroom that has shown antiproliferative activity in various cell lines. However, little is known about the effects of M. esculenta on the cell viability of ovarian cancer. Here, the antiproliferative activity of water, ethanol, and methanol extracts of “grey” and “yellow” M. esculenta fruiting bodies on human ovarian adenocarcinoma cells were investigated. Further, the antioxidant capacity and phenolic content of the mushrooms were evaluated.

Wild “grey” and “yellow” M. esculenta fruiting bodies were harvested in Illinois, USA in April 2019, immediately freeze‐dried, and ground into powders. For antioxidant assays, M. esculenta powder was extracted using 1% HCl in 90% aqueous methanol for 2 h. The antioxidant capacity was evaluated using 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) assays. Both assays revealed significantly (p<0.0001) higher antioxidant capacity (trolox equivalents; TE; μmol/g dry weight) of “grey” compared to “yellow” mushrooms. Specifically, the DPPH assay showed antioxidant activities of 5.35±0.152 and 6.99±0.0190 TE (mean±SEM) for “yellow” and “grey” M. esculenta, respectively. The antioxidant capacity as determined by the ABTS assays was 3.87±0.113 and 9.78±0.112 TE (mean±SEM) for “yellow” and “grey” mushrooms, respectively. The Folin‐Ciocalteu method was used to determine the total phenolic content (gallic acid equivalents; GAE; μmol/g dry weight). In correlation to the DPPH and ABTS results, the phenolic content was significantly higher (p<0.0001) for “grey” compared to “yellow” mushrooms. The phenolic content was determined to be 9.90±0.098 and 12.02±0.316 GAE (mean±SEM), respectively.

For analysis of antiproliferative activity, mushroom powders were extracted thrice for 24 h using water, ethanol, or methanol. Extracts were then air‐dried overnight at 50°C and resuspended in dimethyl sulfoxide (DMSO) to 20 mg/mL. Human ovarian adenocarcinoma (SKOV‐3) cells were then treated with 0.2 mg/mL mushroom extract, 100 μM paclitaxel, or DMSO (vehicle) for 48 h at 37°C, 5% CO2. Cell viability was determined using a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction assay by measuring the absorption of formed formazan at 570 nm. Water and ethanol extracts from both “yellow” and “grey” M. esculenta resulted in significantly reduced cell viability (p<0.05) compared to DMSO. However, methanol extracts failed to show any effect on cell viability. Based on these results, water and ethanol extracts from “yellow” and “grey” M. esculenta significantly lower the cell viability of ovarian adenocarcinoma cells. These mushrooms may, therefore, exhibit antiproliferative potential against ovarian cancer cells.
Role of Histone Deacetylases 5 (HDAC5) in Ang II‐Induced the Early Growth Response Protein‐1 (Egr‐1) Expression and Vascular Hypertrophy
Authors: Truong Vanessa , Jain Ashish , Anand-Srivastava Madhu , Srivastava Ashok
Published: April 2020

Angiotensin II (Ang II), a key vasoactive peptide, is known to play an important role in the pathophysiology of vascular diseases. A heightened activation of Ang II‐induced signaling pathways that promote proliferation, hypertrophy and migration of vascular smooth muscle cells (VSMCs) has been suggested to contribute to vascular dysfunctions. We have shown earlier that Ang II enhances the expression of early growth response protein‐1 (Egr‐1), a zinc transcription factor, which is upregulated in animal models of vascular diseases. Histone deacetylases (HDACs) remove acetyl groups of lysine residues from histone and non‐histone proteins and elevated activation of class IIa HDACs is reported in several vascular pathologies. Recent studies have demonstrated that the phosphorylation and the nuclear export of HDAC5, a member of class IIa HDACs, is associated with vascular remodeling through their ability to interact with transcription factors linked to cell proliferation, migration and hypertrophy. However, the involvement of HDAC5 in Ang II‐induced Egr‐1 expression remain unexplored. Here, we showed that the specific pharmacological inhibition of class IIa HDACs by MC1568 or TMP‐195 as well as siRNA‐mediated silencing of HDAC5 attenuated Ang II induced Egr‐1 expression in VSMCs. In addition, the blockade of the nuclear export of HDAC5 by using nuclear transport inhibitors leptomycin B or KPT‐330 decreased Ang II‐induced Egr‐1 expression. We also demonstrated that siRNA‐induced silencing of HDAC5 or Egr‐1 prevented VSMC hypertrophy in response to Ang II stimulation. In summary, our results show that Ang II‐induced Egr‐1 expression is regulated by the activation of HDAC5 and Ang II‐induced cell hypertrophy is mediated by HDAC5 and Egr‐1.

Support or Funding Information

Supported by the Canadian Institutes of Health Research
Immunohistochemical expression of β‐Catenin associated to bone strain changes in the subchondral bone of mandibular condyle – study in Wistar rats with dental premature contact
Authors: Freire Alexandre Rodrigues , Rossi Ana Cláudia , Okamoto Roberta , Ferreira Beatriz Carmona , Prado Felippe Bevilacqua
Published: April 2020

From the biomechanical concepts regarding the masticatory load distribution, the mandible has a load flow pattern from the teeth to the temporomandibular joint (TMJ). Studies have presented morphological changes in the temporomandibular joint after dental occlusal alterations, such as the premature contacts. Thus, it is important to figure out the bone mechanobiology of the mandibular condyle, involving the relation mechanical stimuli x biological response. The aim of this work was to perform an immunohistochemical analysis of β‐catenin expression associated to a computational simulation by finite element analysis (FEA) to evaluate the relation between the osteocyte mechanosensation and the bone strain. This relation was evaluated in rats with occlusal changes by premature contact. Thirty male rats (Rattus norvegicus albinus), Wistar lineage and 2 months old were used in this study. The animals were divided into two groups: control group (N = 10) and experimental group (N = 20). In the experimental group, premature contact was created by a stainless‐steel fragment, with 0.7 mm thickness, cemented on the first upper molar of all animals. Then, these animals were subdivided into two groups, according to the period with premature contact of 7 days (7D) and 14 days (14D). After euthanasia, the immunohistochemical analysis was performed by indirect immunoperoxidase method for β‐catenin expression, using diaminobenzidine for staining and hematoxylin for counter stain. From the head microCT scans of control and experimental groups, finite element models were created and the FEA was performed for both groups, considering the presence of premature contact in experimental group. In all analyses, the subchondral bone of condyle was analyzed in three regions (medial, central and lateral). The immunohistochemical analysis presented higher expression of β‐catenin in the lateral region in all groups as well as the bone strain in FEA. The experimental group presented higher β‐catenin expression 7D compared to control group. At 14D the β‐catenin expression the bone strain was similar to the control group. Thus, in conclusion, the β‐catenin expression can be associated to the strain sensitivity in the subchondral bone of the mandibular condyle. The premature contact increased the bone strain and the expression of β‐catenin in the subchondral bone.

Support or Funding Information

Brazilian Coordination for the Improvement of Higher Education Personnel (PNPD/CAPES).
Functional Compensation Precedes Recovery of Tissue Mass Following Acute Liver Injury
Authors: Walesky Chad , Kolb Kellie , Winston Carolyn , Henderson Jake , Kruft Benjamin , Fleming Ira , Ko Sungjin , Monga Satdarshan P. , Mueller Florian , Apte Udayan , Shalek Alex , Goessling Wolfram
Published: April 2020

The liver plays a central role in metabolism, protein synthesis and detoxification. It possesses unique regenerative capacity upon injury. While many factors regulating proliferation during liver repair have been identified, the mechanisms by which the injured liver maintains vital functions prior to tissue recovery are unknown. Here, we identify a new phase of functional compensation following acute liver injury that occurs prior to cellular proliferation. By coupling single‐cell RNA‐seq with in situ transcriptional analyses in two independent murine liver injury models, we discover adaptive reprogramming to ensure expression of both injury response and core liver function genes dependent on macrophage‐derived WNT/β‐catenin signaling. Interestingly, transcriptional compensation is most prominent in non‐proliferating cells, clearly delineating two separate phases of liver recovery. Overall, our work describes a new mechanism by which the liver maintains essential physiological functions prior to cellular reconstitution and characterizes macrophage‐derived WNT signals required for this compensation.

Support or Funding Information

C.W. is supported by F32DK111151, the Cholangiocarcinoma Foundation Research Fellowship, and the American Liver Foundation Charles Trey, MD Memorial Post‐doctoral Research Fellowship. W.G. is supported by R01DK090311, R01DK105198, R24OD017870, and the Claudia Adams Barr Program for Excellence in Cancer Research. W.G. is a Pew Scholar in Biomedical Sciences. A.K.S. was supported, in part, by the Searle Scholars Program, the Beckman Young Investigator Program, the Pew‐Stewart Scholars Program for Cancer Research, a Sloan Fellowship in Chemistry, the NIH (1DP2GM1194192RM1HG0061931R01DA046277, and the Bill and Melinda Gates Foundation. U.A. is supported by R01DK098414.
Identification of functional partners of translation Elongation Factor 3 in Saccharomyces cerevisiae
Authors: Manzon Emily R. , Kinzy Terri Goss
Published: April 2020

Fungal infections are responsible for 1.6 million human deaths each year. Additionally, fungal pathogens damage a significant portion of food crops. The number of effective anti‐fungal drugs available is small and many have serious side effects. Furthermore, most of these drugs target aspects of the cell membrane, offering a limited selection as cases of resistance increase. While protein synthesis is not a common focus for drug discovery because of similarities between fungi and host cells, eukaryotic Elongation Factor 3 (eEF3) is highly conserved in fungi but not found in higher eukaryotes. This makes it an excellent target for novel anti‐fungal drugs. eEF3 interacts with the ribosome during the translation elongation step of protein synthesis and is essential for yeast survival. However, little is known about the functional partners of eEF3. We are identifying genes that influence this factor with a screen for suppressors of a conditional eEF3 mutant. The mutant contains two missense mutations F803A R806A in a unique chromodomain insertion in the second ATP binding domain of eEF3 that shows significant interactions with multiple ribosome components by cryo‐EM. Through UV mutagenesis, we randomly generated putative suppressor mutations that restore growth at 37°C to the eEF3 temperature‐sensitive phenotype. Eleven strains showed improved growth rate, reduced temperature sensitivity, and many have altered susceptibility to translation‐inhibiting drugs. Whole genome sequencing of the putative suppressors showed multiple genes potentially involved in eEF3 function. Further analysis of the mutations in these proposed suppressor genes will determine their impact on cell function. These experiments will lay the foundation for the development of a novel class of anti‐fungal therapeutics that target eEF3.

Support or Funding Information

National Institutes of Health RO1 GM57483
Discordance between the neutralization profile of apixaban, betrixaban, edoxaban and rivaroxaban in the clotting assays and anti‐Xa measurements
Authors: Siddiqui Fakiha , Tafur Alfonso , Hoppensteadt Debra , Jeske Walter , Lewis Bruce , Fareed Jawed
Published: April 2020

Introduction

Oral factor Xa inhibitors such as Apixaban, betrixaban, edoxaban and rivaroxaban are widely used for the management of thrombotic and cardiovascular indications. A specific antidote namely andexanet alfa (AA) is approved for the management of bleeding complications with apixaban and rivaroxaban. The dosing regimen of andexanet alfa ranges from 400 – 800 mg bolus followed by 4 – 8 mg/min for up to two hours. This dosing regimen is projected to result in a circulating concentration of 75 – 150 ug/ml. This study is designed to compare the neutralization efficacy of andexanet alfa for various factor Xa inhibitors in clot‐based and anti‐Xa assays.

Materials and Methods

Citrated pooled plasma (CPP) was prepared from individual donors. Active pharmaceutical ingredients (API) version of various factor Xa inhibitors were commercially obtained. Stock solutions of each of the agents were prepared at 1mg/ml and working concentrations at 100 and 10 ug/ml. Each of the factor Xa inhibitor was supplemented in plasma in the concentration range of 0 – 1 ug/ml. The neutralization profile of AA was studied at 100ug/ml and 200ug/ml and saline was used as a control. Clot‐based PT, aPTT and amidolytic anti‐Xa assays. The IC50 for the Xa inhibitory activity were calculated for the control drugs and after supplementation with AA. The neutralization profile in the clot‐based assays was measured in terms of decrease in clotting times.

Results

Apixaban and edoxaban exhibited similar inhibitory profiles in the anti‐Xa assays, where as rivaroxaban and betrixaban exhibited much higher IC50s. AA effectively neutralized the anticoagulant effects of all agents at both the 100 and 200 ug/ml, however the neutralization profile in the clot based was not proportional to the anti‐Xa effects. At 200ug/ml only betrixaban was completely neutralized whereas all the other agents were partially neutralized. The order of neutralization at 200ug/ml was betrixaban > rivaroxaban > apixaban > edoxaban.

Conclusions

These studies show that the neutralization profiles of various Xa agents by AA exhibit differential characteristic response for each of the individual agent. These results indicate that individual dosing based on clinical endpoints for each agent rather than the neutralization of anti‐Xa activity measurement may be more relevant to the clinical outcome.
Effects of aging on substantia nigra dopamine neuron excitability and inhibitory synaptic transmission in mice
Authors: Howell Rebecca Dean , Beckstead Michael
Published: April 2020

The degeneration of substantia nigra dopamine neurons is involved in the etiology for Parkinson’s disease, which is characterized by progressive loss of motor and cognitive functions. The biggest risk factors for Parkinson’s disease are age and sex; most cases occur after age 60 and prevalence is almost two times greater in men than women. While much research in Parkinson’s has focused on the molecular mechanisms underlying dopamine neuron degeneration, very little work has considered the influence of sex and normal aging on the functioning of dopamine neurons, which may contribute to disease risk and presentation. In this work, we studied intrinsic firing properties and synaptic transmission to substantia nigra dopamine neurons with in vivo single unit recordings and whole cell patch clamp recordings in brain slices in male and female C57Bl/6 mice at ages 4, 18 and 24 months. Dopamine neurons recorded from male mice ages 18 months and older showed disruptions in firing activity and an increased propensity to depolarization block compared with younger males, while neurons from females remained largely unchanged across ages. Also, preliminary data suggest enhanced GABAergic transmission in dopamine neurons with age in males; likely a compensatory mechanism to balance the increased dopamine neuron excitability in order to maintain normal motor functioning. Understanding how aging affects dopamine neuron functioning, as well as the balance of excitatory and inhibitory input can help determine areas in the nigrostriatal circuity that are vulnerable to impaired functioning, such as in Parkinson’s disease, as well as therapeutic targets.

Support or Funding Information

NIA grant ‐ R01 AG052606, Presbyterian Health Foundation, Oklahoma Center for Adult Stem Cell Research (OCASCR)
A Comparison of Mass and Potency Equated Bovine and Porcine Heparins
Authors: Krishnappan Sharan , Siddiqui Fakiha , Farooqui Ambar , Iqbal Omer , Kouta Ahmed , Hoppensteadt Debra , Jeske Walter , Walenga Jeanine , Fareed Jawed
Published: April 2020

Introduction

Heparins are extracted from the mast cell rich mammalian tissues such as lung and intestine. While the current source of pharmaceutical grade heparin nationwide is primarily derived from porcine mucosa, bovine mucosal heparins (BMH) are currently being considered by the FDA for re‐introduction in medical and surgical procedures as an alternative to porcine mucosal heparins (PMH). We hypothesized that at USP potency equated levels, bovine and porcine mucosal heparins should be comparable, however at gravimetric concentrations, mass adjusted porcine heparins should exhibit stronger anticoagulant effects in comparison to bovine heparins. The purpose of this study is to compare the clotting profiles and biological properties of mass and USP potency equated BMH and PMH.

Materials and Method

Powdered form of pharmaceutical grade heparins of BMH (140 U/mg) and PMH (190 U/mg) origin were obtained from commercial vendors and stock solution was prepared in saline. Powdered protamine sulfate (USP grade) was reconstituted at 1.0 mg/ml in saline. Whole blood thromboelastographic studies were carried out in TEG 5000 Hemostasis system. Clotting (thrombin time; TT and activated partial thromboplastin time; aPTT) and anti‐protease (anti‐Xa and anti‐IIa) assay were measured by using ACL instrument. Relative neutralization by protamine sulfate was carried out at a fixed concentration of 10 ug/ml.

Results

In whole blood TEG, clotting profile (TT and aPTT) and anti‐protease (anti‐Xa and anti‐IIa), USP potency equated BMH and PMH at concentration range of 0 – 1.0 U/ml showed comparable effects. Protamine sulfate at 10 ug/ml completely neutralized BMH and PMH in in a comparable fashion in clotting profile (TT and aPTT) and anti‐protease (anti‐Xa) while drugs were only partially neutralized in anti‐IIa assay. In mass ratio at 0 – 10 ug/ml, PMH showed strong effects in TEG, anti‐protease (anti‐Xa and anti‐IIa) and clotting profile (TT and aPTT) compare to BMH. BMH and PMH were completely neutralized by protamine sulfate in clotting assay, however in anti‐protease assay BMH was completely neutralized while PMH was only partially neutralized at higher concentrations.

Conclusion

These studies suggest that mass porcine heparin exhibits strong effects compared to mass bovine heparin in gravimetric ratios. However, at potency adjusted levels, bovine and porcine heparins exhibit comparable effects sand can be considered biosimilar. These studies conclude that bovine heparin can be an alternative source of heparin to the prominent porcine heparin.
Development of Phenylazo Piperidine Triazenes for Initiated Release of Aryl Diazoniums in Physiologically Relevant Conditions
Authors: Morrow Madeline Gladys , Davis Garrett J. , Jewett John
Published: April 2020

Triazenes can easily be synthesized from various anilines via diazotization and conjugation with secondary amines, including piperidine. Previous literature has noted that such triazene formation can act as a protecting mechanism for aryl diazoniums that can be subsequently released in highly acidic conditions. Previous work in our own lab pertaining to triazabutadienes (TBDs) has shown analogous reactivity, whereas aryl diazoniums can be released under various conditions including low pH, or by irradiation with UV light. Such initiated release shows potential advantages for studying various biological systems. In this work we show that a class of phenylazo‐piperidine triazenes have similar potential utility to be used as protected diazoniums for biological study. We were able to easily synthesize a small library of triazenes with various electronic properties including those with benzene, p‐nitrobenzene, p‐benzamide, and m‐methoxy substituents, among others. Subsequent studies on these compounds showed that the electronics of the functional groups alters the stability and subsequent reactivity of the respective triazenes allowing for different rates of aryl diazonium release. We have seen that decreasing electron density within the aryl ring leads to more stable, and less reactive triazenes, whereas increasing the electron donating potential of these substituents increases reactivity. Furthermore, we have shown that these triazenes are capable of releasing their diazonium species in neutral pH via irradiation with 350 nm UV light, a phenomenon that has not been previously reported to our knowledge. Lastly, we report the ability for these triazenes to label proteins in situ using UV irradiation and display the utility of a novel coumarin triazine that has analogous reactivity to previously reported coumarin triazabutadienes developed in our lab.

Support or Funding Information

NSF CAREER Award
Protein Tyrosine Phosphatase Type 1B (PTP1B) Contributes To Atherosclerotic Processes By Mechanisms That Involve NADPH‐Oxidase And Calcium Influx
Authors: da Silva Josiane Fernandes , Alves Juliano Vilela , Bolsonni Juliana A. , Costa Rafael M. , Rios Francisco J. , Camargo Livia L. , Montezano Augusto C. , Touyz Rhian M. , Tostes Rita C.
Published: April 2020

PTP1B is a protein tyrosine phosphatase (PTP) widely expressed in different cell type including vascular smooth muscle and endothelial cells. PTP1B activation is linked to downregulation of insulin signalling, impairment of nitric oxide production, induction of endoplasmic reticulum stress and angiogenesis. While inhibition of PTP1B decreases atherosclerotic plaque size and increases systemic levels of interleukin 10 (IL‐10), effects on vascular function are unclear. This study tested the hypothesis that PTP1B inhibition attenuates atherosclerosis‐associated vascular dysfunction by reducing endothelial ROS production.

Methods

Male 12 week‐old apolipoprotein E knockout (apoEKO) and wild type (WT) mice were fed a control diet or a high fat (HF) diet (15% cocoa butter; 0.5% cholesterol; 10% protein; 25% carbohydrate; 65% fat; 9,6 kcal/g) for 8 weeks. Animals were treated with the PTP1B inhibitor claramine, 2 mg/Kg/week, i.p., for 3 weeks. Glucose, cholesterol, low density lipoprotein (LDL) and triglycerides (TG) were measured. In vitro studies were performed in human aortic endothelial cells (HAEC) stimulated with lysophosphatidicholine (10−5 M). ROS production was evaluated by lucigenin and DHE fluorescence. Aortic reactivity was evaluated using isometric transducer myograph. Global PTP irreversible oxidation was determined by immunoprecipitation and western blot. PTP, VCAM, NOX4, NOX5 mRNA expression was determined by qPCR. Phosphorylated protein levels, PTP activity, ROS production and calcium influx were evaluated in stimulated HAECs ± PTP1b inhibitor or vehicle. Experiments were approved by the Ethics Committee on Animal Research of the Ribeirao Preto Medical School, University of Sao Paulo (protocol n°106/2019).

Results

ApoEKO mice showed dyslipidaemia, characterized by increased serum levels of cholesterol (WT = 154.7 ± 2.8 vs. apoEKO = 1668.0 ± 26.7 mg/dL) and LDL (WT = 86.5 ± 1.4 vs. apoEKO = 976.2 ± 17.8 mg/dL). Aortic arch and carotid artery atherosclerotic plaque areas showed higher PTP1B, NOX4 and VCAM gene expression than control areas (p < 0.05). Vasorelaxation to acetylcholine was decreased in apoEKO mice vs. WT mice. Claramine restored this response. In HAECs, LPC stimulated ROS generation by NOX5‐dependent mechanisms. LPC also induced PTP1B expression and activation. PTP1B inhibition in HAECs reduced ROS production and normalized NO production and intracellular calcium responses.

Conclusion

PTP1B contributes to endothelial dysfunction by mechanisms that involve ROS production. PTP1B inhibition may be targeted to reduce vascular injury in atherosclerosis.

Support or Funding Information

FAPESP
In Female Rats, the Sympathoexcitatory and Pressor Actions of Arcuate Nucleus Angiotensin II Vary During the Estrus Cycle and are Mediated by Inhibition of Neuropeptide Y, and Excitation of α‐Melanocyte Stimulating Hormone, Projections to the Hypothalamic Paraventricular Nucleus
Authors: Shi Zhigang , Wong Jennifer , Brooks Virginia L.
Published: April 2020

In male rats, nanoinjections of Angiotensin II (AngII) into the arcuate nucleus (ArcN) increases sympathetic nerve activity (SNA) by suppressing tonic sympathoinhibitory Neuropeptide Y (NPY) inputs, and activating excitatory pro‐opiomelanocortin (POMC) inputs that release alpha melanocyte stimulating hormone (α‐MSH), into the hypothalamic paraventricular nucleus (PVN). In female rats, ArcN AngII AT1a receptor (AT1aR) expression is generally higher than in males, but varies with the estrus cycle (highest during estrus; lowest during proestrus). However, whether ArcN AngII increases SNA in females, or if the response varies during the cycle, is unknown. Therefore, we tested bilateral ArcN AngII injections (30 pmol in 30 nL) in α‐chloralose anesthetized female rats throughout the reproductive cycle, and found that 90 min after ArcN AngII injections, LSNA increased (P<0.05) in rats during estrus (by 72±12 %; n=6) and diestrus (by 40±9 %; n=4), but not proestrus (−1±8 %; n=5). ArcN AngII also induced an initial rapid rise (P<0.05) in mean arterial pressure (MAP) in all groups (estrus: 13±2 mmHg; diestrus: 11±3 mmHg; proestrus: 13±3 mmHg), but the increase was sustained only in estrus rats (by 12±2 mmHg; P<0.05). We next tested if ArcN AngII suppresses inhibitory NPY inputs by determining if blockade of PVN NPY Y1 receptors (Y1R) with bilateral nanoinjections of BIBO3304 (60 pmol/60nL) elicits smaller increases in MAP and LSNA 60 min after nanoinjection of AngII into the ArcN. As previously reported, PVN injections of BIBO3304 (without ArcN AngII; n=3) increased (P<0.05) LSNA (by 34±6 %) and MAP (by 10±1 mmHg). However, the increases (P<0.05) in LSNA (to 93±16 %) and MAP (by 19±1 mmHg) (n=4) following ArcN AngII and PVN BIBO3304 were the same as ArcN AngII followed by PVN artificial cerebrospinal fluid (n=3; LSNA by 107±30 % and MAP by 17±4 mmHg). Thus, ArcN AngII markedly attenuated the pressor and sympathoexcitatory effects of blockade of PVN Y1R, indicating that AngII increases LSNA and MAP in part by inhibition of NPY projections to the PVN. Finally, because suppression of tonic NPY inhibition of PVN presympathetic neurons permits increased activation of PVN α‐MSH receptors (MC4R), we tested if blockade of PVN MC4R with SHU9119 decreases LSNA and MAP 90 min after ArcN AngII (n=4). Indeed, bilateral PVN SHU9119 decreased (P<0.05) LSNA (by 30±10 %) and MAP (by 10±1 mmHg). In conclusion, in females as in males, ArcN AngII increases SNA and MAP by inhibiting NPY inputs and increasing α‐MSH inputs into the PVN. However, these effects vary during the estrus cycle in parallel to AngII AT1aR expression: highest during estrus and undetectable during proestrus.

Support or Funding Information

HL128181
Prior Ablation of the Splanchnic Sympathetic Nerves Increases Plasma Inflammatory Cytokine Levels and Suppresses Bacteremia in Response to Systemically Administered E. coli in Sheep
Authors: McKinley Michael J. , Almeida Kaylani , Bedoui Sammy , Farmer David G.S. , Hocking Dianna , Lankadeva Yugeesh , Martelli Davide , May Clive N. , Robins-Browne Roy M. , McAllen Robin M.
Published: April 2020

The splanchnic sympathetic nerves exert a powerful anti‐inflammatory action in response to systemic administration of lipopolysaccharide in rats (Martelli et al. J. Physiol. 592: 1677–1686, 2014). However, it is not known how this endogenous anti‐inflammatory influence of the splanchnic nerve affects the body’s ability to fight infection. The aim of this study was to determine if and how the endogenous reflex activation of the splanchnic anti‐inflammatory pathway affects blood cytokine levels and the disease course of bacteremia after intravenous infusion of live E. coli into a conscious large animal (sheep). One month prior to infection, the greater splanchnic nerve was cut bilaterally (SplancX, n = 8) or sham surgery (Sham, n = 7) performed in anesthetized sheep (isoflurane/O2). After 1 month, these conscious sheep were infused with live E. coli (2.8 × 109 CFU in 30 min) into a jugular vein. Blood samples were obtained at 1.5, 3, 6, 24 and 48 h after bacterial injection. As expected, i.v. infusion of E. coli caused systemic bacteremia with high blood counts of E. coli [3.02 ± 0.64 Log10(CFU/ml + 1), mean ± sem] in 5 of 7 sham sheep. By contrast, no significant bacteremia was observed in any of the 8 SplancX sheep (0.18 ± 0.18 Log10[CFU/ml + 1], p < 0.01, c.f. Sham sheep). Plasma levels of tumour necrosis factor‐α peaked after 1.5 h, and were significantly greater in SplancX vs Sham sheep (2.4 ± 0.6 vs 1.5 ± 0.2 ng/ml, p < 0.05) at this time. Plasma levels of another pro‐inflammatory cytokine, interleukin‐6, peaked at 6h and were higher in SplancX vs Sham sheep (7.4 ± 0.6 vs 2.4 ± 0.6 ng/ml, p < 0.01). Although plasma levels of the anti‐inflammatory cytokine interleukin‐10 increased in both groups, they were lower in SplancX vs Sham sheep from 1.5 h to 24 h (2.9 ± 0.4 vs 6.7 ± 1.1 ng/ml at 24 h, p < 0.01). All plasma cytokine levels measured had returned to baseline by 48 h. Plasma cortisol levels were higher (p<0.01) in SplancX sheep only at 6 h (298 ± 29 vs 138 ± 37 ng/ml), while the fever was less (p < 0.05) in SplancX at 6 h (40.3 ± 0.3 vs 40.8 ± 0.3°C). The greater pro‐inflammatory response in splanchnic denervated animals most likely contributes to their enhanced ability to clear bacteremia. The present findings demonstrate the powerful consequences of neural control of inflammation in the context of an acute systemic infection.

Support or Funding Information

Supported by the National Heart Foundation of Australia
A reliable LC‐MS/MS method for targeted analysis of amino acid metabolome: Method validation and application to studies on amino acid dynamics during tumor progression
Authors: Liu Zhenzhen , Tu Mei-Juan , Zhang Chao , Jilek Joseph Lionel , Zhang Qian-Yu , Yu Ai-Ming
Published: April 2020

A simple and fast LC‐MS/MS method was developed and validated for targeted quantification of 20 proteinogenic L‐amino acids (AAs) in mouse plasma. Chromatographic separation was achieved on an Intrada Amino Acid column within 13 min via gradient elution with an aqueous solution containing 100 mM ammonium formate and an organic mobile phase containing acetonitrile, water and formic acid (v: v: v = 95: 5: 0.3), at the flow rate of 0.6 mL/min. Individual AAs and corresponding stable‐isotope‐labeled AA internal standards were analyzed by multiple reaction monitoring (MRM) in positive ion mode under optimized conditions. Method validation consisted of linearity, sensitivity, accuracy and precision, recovery, matrix effect, and stability, and the results demonstrated this method as a specific, accurate, and reliable assay. This LC‐MS/MS method was thus utilized to examine the dynamics of plasma AA metabolome during tumor progression in an orthotropic hepatocellular carcinoma (HCC) xenograft mouse model. The results reveal a significantly lower Arg concentration as well as higher Ala and Thr levels during HCC progression. These findings support the utilities of this LC‐MS/MS method and the promise of specific AAs as possible biomarkers for HCC.

Support or Funding Information

National Cancer Institute (grant No. R01CA225958)

National Institute of General Medical Sciences (R01GM113888)