bluuebunny/crossref_metadata_2025 huggingface dataset:
Noncompetitive allosteric antagonism of death receptor 5 by an affibody discovered via yeast display
Authors: Vunnam Nagamani , Szymonski Sophia , Petra Hirsova , Gores Gregory James , Hackel Benjamin , Sachs Jonathan
Published: April 2020

Fatty acid‐induced upregulation of death receptor 5 (DR5) and its cognate ligand, tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL), promotes hepatocyte lipoapoptosis, which is a key mechanism in the progression of fatty liver disease. Accordingly, inhibition of DR5 signaling represents an attractive strategy for treating fatty liver disease. Ligand competition is prevalent in tumor necrosis factor receptor antagonism, but recent studies suggest a compelling alternative: non‐competitive inhibition through perturbation of receptor conformation. We used a yeast‐displayed combinatorial library to identify a synthetic affibody that specifically binds DR5. Biophysical and biochemical studies show that the affibody neither blocks TRAIL binding nor prevents the receptor‐receptor interaction. Live‐cell fluorescence lifetime measurements indicate that the affibody induces a conformational change in transmembrane dimers of DR5 and favors an inactive state of the receptor. The affibody inhibits apoptosis in TRAIL‐ or palmitate‐treated Huh‐7 cells, an in vitro model of fatty liver disease. Thus, this lead affibody serves as a potential drug candidate, with a unique mechanism of action, for fatty liver disease.

Support or Funding Information

National Institutes of Health Grants R01EB023339, R01DK41876, R35GM131814 and R01EB028274.
A Systematic Review of Spatial Ability & Anatomy Performance: It’s time for a metaanalysis
Authors: Roach Victoria A. , Mi Misa , Mussell Jason C. , Van Nuland Sonya E. , Lufler Rebecca S. , DeVeau Kathryn M. , Dunham Stacey M. , Husmann Polly R. , Herriott Hannah L. , Edwards Danielle N. , Doubleday Alison F. , Wilson Brittany M. , Wilson Adam B.
Published: April 2020

Rationale

Spatial ability has been defined theoretically as ‘the ability to make use of simulated mental imagery (often in conjunction with currently perceived images) to solve problems'. Despite being among the most investigated factors of human cognitive functioning, spatial ability has been relegated to a secondary status within human intelligence research due to inconsistent evidence supporting spatial skills as predictors for educational success. Recently, however, interest in spatial ability within the anatomical community has grown, due to correlational evidence associating spatial aptitude with educational performance in the areas of science, technology, engineering, and mathematics. As a result, a substantial volume of contemporary research has focused on further uncovering the role that spatial ability plays in anatomical knowledge acquisition and retention. As such, this current study utilized a systematic review (to be followed by a meta‐analysis) to identify the extent to which spatial ability affects performance in anatomy.

Methods

Relevant studies published within the past 50 years (2019–1969) were searched for across eight databases. Predetermined eligibility criteria were applied to the screening of titles and abstracts to discern their appropriateness for study inclusion. Study outcomes were first thematically analyzed (reported herein) and will be followed by a true meta‐analysis.

Results

A total of 2,458 records were screened, 59 underwent full‐text review, and 21 studies (k) were included for final analysis. Of these studies, 57% (12 of 21) were from North America, most (15 of 21) studied graduate/professional students, and all were published in 2000 or later, except for one study from 1985. Most studies (71%; 15 of 21) evaluated the association between spatial ability and anatomy performance using the Mental Rotations Test (k=14) and some form of a practical assessment of anatomical knowledge (k=15). Of the 15 correlational studies 6, 7, and 2 reported significant, non‐significant, and inconclusive findings respectively. The prevalence of conflicting studies further necessitates the need for a meta‐analysis, which is currently underway.

Conclusions

High spatial ability is often associated with better scores in anatomy, but some studies show no association. In the absence of a meta‐analysis, it is challenging to interpret the literature to determine the significance of practical implications, such as providing students with different study techniques or presentation modalities depending on their level of spatial ability. Only a meta‐analysis will better inform our understanding of the current literature.
PVN‐projecting NTS neurons receive both direct and indirect inputs from solitary tract afferents
Authors: Fawley Jessica , Hegarty Deborah , Aicher Sue , Doyle Mark , Beaumont Eric , Andresen Michael
Published: April 2020

Cranial vagal afferents first synapse on second‐order neurons in the brainstem nucleus of the solitary tract (NTS). Synaptic transmission in the NTS regulates a wide diversity of homeostatic functions and NTS neurons project widely to other brain regions, including the paraventricular nucleus of the hypothalamus (PVN). The PVN itself is also highly heterogeneous and its neurons both receive and send projections to many other brain areas. Expression of the Transient Receptor Potential Vanilloid receptor (TRPV1) distinguishes solitary tract (ST) afferents as either C‐fibers (TRPV1+) or A‐fibers (TRPV1‐). Sensitivity to resiniferatoxin (RTX), an agonist to the non‐selective cation channel TRPV1, differentiates the two. It is unknown if TRPV1+ or TRPV1− afferents selectively innervate PVN‐projecting neurons or whether every contact on a single PVN neuron is from a segregated pathway to the PVN (i.e., information from A‐ and C‐fibers remains on a labelled line). To identify NTS neurons that specifically project to the PVN, we injected the fluorescent retrograde tracer rhodamine into the PVN. In rhodamine‐positive NTS neurons, responses to ST‐shocks elicited EPSCs with latencies that had either low jitters (<200 μM) that identified direct afferent connections, or high jitter (>200 μs) that identified indirect, higher order inputs. Extensive recruitment curves identified the number and synaptic order of each input. Most neurons received a mix of 2nd and higher order inputs. A minority of cells received only higher‐order contacts or only direct inputs. The amplitude variance of evoked EPSCs for higher order inputs was significantly greater and had more failures than that of second order inputs. Asynchronous release from indirect inputs was often absent. A majority of afferents were TRPV1+ and all neurons had an all‐or‐none response to RTX. These preliminary results suggest that all inputs on PVN‐projecting NTS neurons, including neurons that receive both second and higher inputs, are either all TRPV1+ or all TRPV1−.

Support or Funding Information

HL133505 and HL141560
CYP3A7 drug‐hormone Interactions in the Neonate: Adrenal Insufficiency and Low Birthweight in the HIV infected Neonate caused by Inhibition of CYP3A7 DHEA‐S Oxidation by Ritonavir
Authors: Lampe Jed Noah , Kandel Sylvie Eliane
Published: April 2020

In the neonate and developing infant, CYP3A7 is the predominant CYP3A isoform and is responsible for the majority of metabolism of CYP3A substrates. However, its primary function is the 16α‐hydroxylation of the endogenous hormone DHEA‐S, which is important for maintenance of hormone homeostasis in the adrenal gland and production of estriol during the third trimester of pregnancy. Low maternal estriol levels have been reported to lead to low birth weight and premature birth. It has been previously reported that cohorts of HIV infected mothers and their newborns on a combined therapeutic regimen of Lopinavir and Ritonavir have increased adrenal insufficiency and low birth weight, as well as substantially elevated levels of DHEA‐S, leading to adverse outcomes in this fragile patient group. Since Ritonavir is a known potent inhibitor of CYP3A4, we hypothesized that it might also be an inhibitor of CYP3A7‐mediated DHEA‐S oxidation and that this inhibition might be responsible for the adrenal insufficiency and low birth weight associated with Lopinavir‐Ritonavir treatment. In order to test this hypothesis, we employed both recombinant CYP enzyme and neonatal human liver microsomes (nHLMs). We observed significant inhibition of CYP3A7 DHEA‐S hydroxylation activity in the presence of Ritonavir with an IC50 of 0.05 μM. Furthermore, Ritonavir displayed time‐dependent inhibition of DHEA‐S oxidation, with a Ki of 0.336 μM and a Kinact of 0.1077 min−1, suggesting that it may be a mechanism based inactivator of CYP3A7. Lopinavir was also capable of inhibiting CYP3A7, albeit to a much lesser extent than Ritonavir with an IC50 of 5.88 μM for DHEA‐S 16α‐hydroxylation, and without the associated time‐dependency. Interestingly, we observed that Lopinavir alone had a lower intrinsic clearance (Clint) rate in nHLMs compared to adult HLMs, suggesting that a pharmacokinetic enhancer adjuvant may not be necessary in this patient population in order to slow drug clearance and maintain efficacy. At a minimum, our results suggest that closer monitoring of the effective dose of the pharmacokinetic enhancer in neonatal HIV therapy may be required in order to prevent significant inhibition of CYP3A7 DHEA‐S oxidation and the associated adverse effects.

Support or Funding Information

This work was supported by the National Institutes of Health, Institute of General Medical Sciences [Grant R01 GM128508‐03] to JNL.
Identification and molecular characterization of a novel non‐inhibitory isoform of human neuroserpin gene
Authors: Kalam Sayeed ur Rehman Abul , Jairajpuri Mohamad Aman
Published: April 2020

Neuroserpin is linked to conditions like Alzheimer’s disease, dementia, epilepsy, and FENIB. It is a member of serpin superfamily and inhibits proteases like tPA, trypsin, uPA, plasmin, and NGF‐γ. Apart from the inhibitory roles, various non‐inhibitory functions of neuroserpin are also reported.

Alternative splicing leads to the generation of multiple isoforms having similar, different or opposing functions. It is a highly regulated process and is linked to various disease conditions. Alternative splicing of human neuroserpin gene may generate multiple isoforms. However, only two transcripts of neuroserpin have been reported with variations in the promoter regions.

Using a combined approach of bioinformatics and molecular biology, we have identified a novel alternatively spliced transcript of the human neuroserpin gene in the brain and liver. This novel transcript codes for a truncated protein and lacks the reactive center loop which is responsible for the inhibitory action of neuroserpin. The existence of novel transcript was confirmed using RT‐PCR, DNA sequencing and qRT‐PCR. Structural and functional characterization of novel isoform was performed using recombinant protein expressed in BL21(DE3) cells. UV‐visible, fluorescence and CD spectroscopy was used to perform the structural analysis while inhibition assays using tPA and plasmin confirmed the non‐inhibitory nature of the novel isoform. The truncated isoform identified in this study is expected to be involved in crucial non‐inhibitory roles in the cell.
Connexin 43 Regulates Multiple Aspects of Chick Cranial Neural Crest Cell Development
Authors: Jourdeuil Karyn , Taneyhill Lisa A.
Published: April 2020

Connexins are transmembrane proteins that cluster to form intercellular channels called gap junctions, which allow direct diffusion of small molecules and ions between adjacent cells. Connexin 43 (Cx43) is one of the predominant components of gap junctions and is expressed in multiple organisms and their tissues. To date, however, very little is known about the role of gap junctions, and specifically Cx43, in the neural crest. Our prior immunohistochemical analyses revealed that Cx43 is expressed in premigratory cranial neural crest cells both prior to and during their epithelial‐to‐mesenchymal transition (EMT), and in migratory neural crest cells. Given the robust expression of Cx43 in premigratory neural crest cells, we sought to elucidate the role of Cx43, and gap junctions in particular, during neural crest cell EMT. To this end, we evaluated the presence of gap junctions in the neural crest and performed perturbation experiments to alter the levels of Cx43 in both premigratory and migratory neural crest cells. Our fixed and live imaging results reveal that gap junction formation occurs within both the premigratory and migratory neural crest cell populations. Moreover, morpholino‐mediated reduction in Cx43 protein levels prevents neural crest cells from forming functional gap junctions, as assessed by live imaging of transfer of the gap junction‐specific dye Calcein in the embryo. Additionally, depletion of Cx43 from premigratory neural crest cells reduces the size of the premigratory neural crest cell domain and also delays the emergence of neural crest cells from the dorsal neural tube. This delayed neural tube exit phenotype can be rescued by introduction of full‐length rat Cx43 that is refractory to the morpholino. These data suggest that gap junctions, and Cx43 in particular, are involved in early neural crest cell specification and in the emergence of neural crest cells from the neural tube.

Support or Funding Information

The work was supported by an American Association for Anatomy Postdoctoral Fellowship to K.J. and grants to L.A.T. (NIH R01DE024217, American Cancer Society RSG‐15‐023‐01‐CSM).
Interleukin‐16 Signaling Through the CD9 Receptor Initates Rearrangement of the Actin Cytoskeleton
Authors: Charley Lindsey Allyce , McKinney Catie , Blake David , Fenster Steven D.
Published: April 2020

CD9 is a tetraspanin membrane protein that plays critical roles in various cellular processes including cellular proliferation, activation, intracellular signaling, survival, invasion, migration, cell adhesion, and cytoskeletal rearrangement. All major subsets of leukocytes, as well as some endothelial cells, express the CD9 receptor. Activation of CD9 occurs through interactions with a variety of cell surface molecules including integrins and other adhesion molecules. Recent evidence has indicated that the cytokine, Interleukin‐16 (IL‐16), can also serve as a ligand for activation of the CD9 receptor. IL‐16 is best known as an immunomodulatory, pro‐inflammatory cytokine that aids in CD4+ cell recruitment and stimulation at sites of inflammation through signaling via the CD4 receptor. However, CD9 has been implicated as an alternative receptor in several cell types lacking the CD4 receptor. For example, recent studies have established that cells deficient in CD4 are reactive to IL‐16 including increased cytokine production, cell migration, and neural outgrowth. Recently, we showed that in a human lung cancer epithelium cell line (A549 cells), CD9 is required for cell migration specifically towards IL‐16 providing compelling evidence that CD9 is an alternate receptor for IL‐16. In this study, we used A549 cells either expressing CD9 (CD9+) or lacking CD9 (CD9−) due to genetic ablation via CRISPR/Cas technology to examine the effects on cytoskeletal rearrangement upon activation following incubation with recombinant IL‐16. Our preliminary evidence indicates that in A549 cells, IL‐16 treatment initiates robust rearrangement of both the actin cytoskeleton and associated proteins such as vinculin and talin. Further studies will examine the differences in IL‐16‐mediated cytoskeleton reorganization between CD9+ and CD9− cells.

Support or Funding Information

NIH‐MARC U*STAR award number T34GM092711

BLaST Undergraduate Research Experience

CO‐WYAMP of NSF LSAMP: Mini‐grant
Targeting Doublecortin‐like Kinase 1 (DCLK1) in Colon Cancer: An In‐silico Approach
Authors: Parsai Janhvi , Crockett Cooper , Kimball Hunter , Raval Nipam
Published: April 2020

Cancer stem cells (CSCs) are proposed to be a contributing factor in colorectal cancer (CRC) recurrence and chemo‐resistance, as a result of increased expression of ABC transporters and their innate ability to self‐renew and enter a quiescent state. Current standard therapies are ineffective at eliminating these CSC populations, hence there is a need to identify novel agents to effectively inhibit their growth. Multiple surface markers have been identified for CRC‐CSCs, though few have shown promise as a clinical target. Recently, doublecortin‐like kinase 1 (DCLK1) was established as a CSC marker in CRC and pancreatic cancers. The N‐terminal doublecortin domains(DCX) bind microtubules and regulate their polymerization, while the C‐terminal serine/threonine‐protein kinase domain is involved in cellular signaling. DCLK1 is a marker of Tuft cells in normal intestinal crypts, but in cancer is found to be highly expressed in CSC populations that play a critical role in tumor progression, based on lineage tracing experiments. Specific ablation of DCLK1+ stem cells causes marked regression of polyps without apparent damage to the normal intestine, distinguishing DCLK1 as a CSC marker. Recently, marmelin, a phytochemical from Aegle marmelos, has been shown to target DCLK1 and reduce CRC tumor growth in vivo. Hence, DCLK1 represents an attractive target for CSC‐targeted drug discovery. The present study is aimed to identify small molecule inhibitors of DCLK by using in silico molecular docking‐based screening. For this, we tested a library of FDA‐approved drugs and kinase inhibitors using Autodock Vina software. Compounds were assessed on their ability to bind within the ligand binding site of the C‐terminal kinase domain. The compounds are ranked‐based on their binding energies and hydrogen bonding patterns.

Support or Funding Information

The Olathe North MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine the structure‐function relationship in this project.
Supplemental treatment options for diabetes: how flavanol metabolites improve β‐cell function
Authors: Lloyd Trevor , Griffin Laura , Krueger Emily , Beales Joseph , Barlow Andrew , Sheets Jared , Ekpo Idongesit , Ross Mimi , Chandra Preeti , Rathore Atul , Kay Colin , Neilson Andrew , Tessem Jeffery Sivert
Published: April 2020

Diabetes is one of the fastest growing non‐infectious diseases in the world. Current treatments are composed of pharmaceutical agents that enhance insulin sensitivity and eventual insulin monotherapy. Type 2 diabetes is characterized by insulin insensitivity of peripheral tissue, glucose intolerance, and β‐cell dysfunction. Dietary interventions may benefit patients with diabetes, and various plant derived flavonoids have been shown to exert anti‐diabetic effects. While these flavonoids are large, difficult to absorb, and rarely found in circulation, gut bacteria metabolize these into smaller metabolites which can be observed in circulation. We hypothesize that these gut bacteria derived flavanoid metabolites are absorbed and have direct effects on β‐cell function. Male outbred wistar rats were fed one of three diets in the presence or absence of antibiotic treatment: standard diet, standard diet supplemented with catechin hydrate and epicatechin, or standard diet supplemented with grape seed extract. Total urine was collected from the animals (representing the total amount of absorbed metabolites), then metabolites were extracted and reconstituted in water. Here we present data regarding the in vitro effects of these absorbed gut bacteria derived flavanoids on INS‐1 832/13 β‐cell insulin secretion and proliferation. This study sheds further light on the potential ability of flavanoids and their gut bacteria derived metabolites to enhance functional β‐cell mass.
Glypican‐1 and Remodeling Cardiac Hypertrophy.
Authors: Hansen Madeline , Garcia Roxanne , Dull Randal O. , Carvalho-de-Souza Joao L. , Chignalia Andreia Z.
Published: April 2020

Heart failure impacts about 5.7 million people in the U.S. alone and the clinical signs often arise from changes in heart function. Cardiac hypertrophy is a known contributor to impaired cardiac contractility and ejection fraction. The heart deals with a working relationship with volume and pressure and thus mechanosensitive proteins may play a role on heart function. This study investigates the role of heparan sulfate proteoglycans, specifically syndecan1 (Sdc1) and glypican1 (Gpc1) on heart function and hypertrophy. We investigated if global deletion of Gpc1 and Sdc1 in mice alters heart histological features and thus cardiac function. Global male knockout mice for Sdc1 (Sdc1KO) and Gpc1 (Gpc1KO) were used and compared to its respective controls C57B6 and CD1 (7 weeks old). Left ventricular posterior wall thickness at end diastole (LVPWd), interventricular septal thickness at end of diastole (IVSTd) and left ventricular internal diameter in diastole (LVIDd) and myocyte cross‐sectional area in the left ventricle were assessed by transmission microscopy using hematoxylin & eosin (H&E) staining. Collagen deposition was assessed by Masson’s Trichrome staining. No differences in heart dimensions (LVPWd, IVSTd, LVIDd) were found between C57B6 and Sdc1KO. IVSTd and LVPWd was increased in Gpc1KO compared to CD1 (IVSTd: 1.15 mm for Gpc1KO and 1.03 mm for CD1; LVPWd: 0.75 mm for Gpc1KO and 0.64 mm for CD1). Both knock out lines showed decreased myocyte cross‐sectional area in the left ventricle size compared to its control (203.86 μm2 for Gpc1KO, 248.43 μm2 for CD1, 194.64 μm2 for Sdc1KO, and 239.02 μm2 for C57B6). Blind scoring of collagen staining showed that there was not a significant difference in collagen deposition between Gpc1KO and CD1 (1.14 for Gpc1KO and 0.99 for CD1). Taking together, these findings indicate that Sdc1KO and Gpc1KO show alterations in cardiac histological features. Our data raises the possibility of heparan sulfate proteoglycans being involved in cardiomyocyte proliferation or development. Particularly, Gpc1KO show the unique characteristic of having a hypertrophic heart without collagen deposition. Heparan sulfate proteoglycans may play a key role in cardiac function and we speculate that they may be involved in heart disease progression.

Support or Funding Information

Department of Anesthesiology, College of Medicine, University of Arizona, Tucson
Proteomics Analysis of a Phage‐Bacterium Interaction Reveals Both Hijacking and Suppression of Host Metabolic Processes
Authors: Okekeogbu Ikenna Ochiagha , Aryal Uma , Clase Kari
Published: April 2020

The potential for phages to serve as both therapeutic agents and in broad applications ranging from food bio‐preservation, wastewater treatment, and disease diagnosis, has led to renewed interest in the study of phage‐bacteria interactions. Phages attack their hosts by hijacking its molecular machinery and using it for its own replication, subsequently shutting off the host macromolecular synthesis. Not much is known about how this mechanism occurs, however, especially at the translational level. In this study, label‐free tandem mass spectrometry based quantitative proteomics and qPCR assay of phage Ochi17‐infected Mycobacterium smegmatis suggested that shutoff of the host macromolecular synthesis also involves suppression of its resistance mechanisms. A total of 2,188 proteins were identified in the analysis, with 299 significantly up‐regulated proteins and 135 down‐regulated proteins. The results indicated phage Ochi17 DNA integration into the host’s genome, and a consequent hijack and suppression of the host’s energy metabolic processes and antibiotic resistance mechanisms as seen in the significant enrichment of proteins involved in homologous recombination, transcriptional factors, ribosomal proteins, amino acid (arginine, proline, and histidine) metabolism, and oxidoreductase activities. Majority of the proteins were found to be part of the host’s energy metabolic processes. With these results, we propose that phages counter their host defensive response by suppressing these responses in addition to evolving new attack strategies. We also reported the expression of 59 phage proteins (54%) expressed during the interaction. This study provides the first global proteome investigation and characterization of any phage‐infected bacteria with respect to the host bacterium response. These Results would serve as a rich source for further molecular studies of phage‐bacteria interactions in elucidating contributions of individual proteins, and in engineering phages that can overcome various defense strategies employed by host bacteria.

Support or Funding Information

This work was supported by the Biotechnology Innovation and Regulatory Science (BIRS) Center, Purdue University.
The Effects of 5‐Azacytidine on HLA‐ABC, HLA‐E, HLA‐G and HLA‐A Expression
Authors: Hansen Adam , O'Donnell Robert W.
Published: April 2020

Epigenetic modifiers, such as the DNA methylation inhibitor 5‐Azacytidine (5‐AzaC), have been shown to increase human leukocyte antigen (HLA) expression. The expression of HLA is required for a T‐cell response to detect tumor cells. A lack of HLA expression allows tumor cells to escape immune detection. It has been previously shown that 5‐AzaC is able to upregulate HLA‐ABC expression in the cell line MDA‐MB‐435. However, other HLA proteins like HLA‐E and HLA‐G may be concurrently upregulated, which would have negative implications on tumor immunity. One flask of MDA‐MB‐435 cells was treated for 48 hours with 5‐AzaC at 0.0025 mg/mL and another was left untreated. The cells were harvested and incubated with control antibodies or the experimental antibodies, Anti‐HLA‐ABC, Anti‐HLA‐G, and Anti‐HLA‐E and analyzed via flow cytometry. Preliminary experiments testing for the expression of HLA‐E and HLA‐G have both demonstrated a slight increase in their expression compared to the untreated cells. Although minor, the increase in HLA‐E and HLA‐G expression may have negative implications on tumor survival and pathogenesis. Further experimentation on HLA‐E and HLA‐G expression, as well as HLA‐A expression alone, will be conducted. Understanding individual HLA upregulation may prove to be beneficial and applicable to cancer immunotherapy, an expanding field of improved cancer treatments.
Critical role of arginase 2 in obesity‐induced metabolic dysregulation in female mice: Implication of macrophage inflammatory response
Authors: Eldahshan Wael , Atawia Reem T. , Meghil Mohamed M. , Fouda Abdelrahman Y. , Cutler Christopher W. , Caldwell Ruth B. , Caldwell Robert W.
Published: April 2020

We have previously shown that deletion of the mitochondrial isoform of the ureahydrolase enzyme (A2) protected against obesity‐induced metabolic dysregulation in “male” mice fed high fat high sucrose diet (HFHS) for 16 weeks. Additionally, A2 expression was upregulated 1.5‐fold in visceral adipose tissue (VAT) of WT "male" mice fed HFHS vs WT fed normal chow diet (ND). Interestingly, A2 expression was elevated 3‐fold in VAT of WT HFHS "female" mice vs WT ND. In the current study, we determined the consequences of global A2 deletion in “female” mice on the same diet protocol. Body weight was measured weekly, adiposity was measured using nuclear magnetic resonance and metabolic activity was assessed using indirect calorimetry. Increases in body weight in WT HFHS female mice, VAT weight and total adiposity were abrogated in A2−/− HFHS mice by 24, 84 and 54%, respectively (p<0.05). Glucose tolerance tests showed a significantly reduced area under the curve in A2−/− HFHS mice vs WT (p<0.05). The leaner phenotype observed in A2−/− mice was in line with the improved metabolic activity as evidenced by increased energy expenditure, volume of O2 consumed and CO2 produced in A2−/− mice fed HFHS vs WT HFHS. Obesity‐induced VAT inflammation and vascular rarefaction contribute to unhealthy expansion of adipose tissue resulting in disrupted metabolic and endocrine functions. VAT sections from WT HFHS stained with isolectin B4 revealed reduced vascular density which was preserved in A2−/− HFHS. Flowcytometry analysis of stromal vascular fraction isolated from VAT of WT HFHS mice showed an increase in proinflammatory M1 macrophages (F4/80+CD11c+) (p=0.06) and a significant decrease in antiinflammatory M2 macrophages (F4/80+CD206+) (p<0.05), the latter was not affected in male mice. These effects were blunted in A2−/− HFHS female mice, reducing the M1/M2 ratio towards a favorable, less inflammatory state. Given the role of A2 in polarizing macrophages to an inflammatory state in vivo, we wished to determine the specific effect of macrophage A2 on inflammatory responses. Peritoneal macrophages from WT and A2−/− female mice were isolated, cultured in vitro and treated with 100 ng/ml lipopolysaccharide (LPS) or vehicle for 24 hr. WT macrophages exposed to LPS showed an activated inflammasome pathway (increased expressions of NLRP3 and pro‐IL‐1β) compared to control group (p<0.05). These effects of LPS were blunted in isolated A2−/− macrophages, indicating that A2 promotes inflammatory macrophage responses in female mice. Collectively, our data show that A2 is a critical mediator of obesity‐induced metabolic dysregulation, VAT vascular rarefaction and increased macrophage inflammatory responses in female mice.

Support or Funding Information

R01 HL070215, R01 EY01176 & AHA17PRE33660321
Effects of Vacuolar H+‐ATPase Inhibition and the a2 Isoform on Secretion and Activation of Cathepsin B in MDA‐MB231 Breast Cancer Cells
Authors: Henry Abhijeet Anil
Published: April 2020

V‐ATPases are multi‐subunit H+ pumps that acidify intracellular compartments and are important for immune functionality, synaptic signaling, and other processes. They are at the cell surface of the highly metastatic breast cancer cell line MDA‐MB231 and may contribute to its metastatic phenotype. We hypothesize that V‐ATPases create a low pH environment that promotes tumor cell invasion by activating secreted cathepsins. Studies indicate that cathepsins secreted by cancer cells are involved in metastasis. Cathepsins cleave extracellular matrix proteins and activate other proteases, facilitating tumor invasion. We are interested in a connection between cell surface V‐ATPases, activation of secreted cathepsins and the metastatic phenotype of MDA‐MB231 cells. Previous data from our lab indicates that inhibition of V‐ATPases by a general inhibitor reduced the activity of secreted cathepsin B, but did not cathepsin L. However, although there was a trend, difference in activity for secreted cathepsin B after V‐ATPase inhibition was not statistically significant. Our current data builds on that trend to show a significant difference in the activity of secreted cathepsin B. To investigate the role of V‐ATPases in the activation of cathepsins further we are using CRISPR/Cas9 to knockout the gene for the V‐ATPase a2 isoform of subunit “a”. Published data indicates that the a3 and a4 isoforms are found in V‐ATPases at the cell surface of MDA‐MB231 cells. Additionally, it was found that the a1 and a2 isoforms are not present on the cell surface, thus we hypothesize that the a2 isoform knockout will not affect the activity of secreted cathepsin B, but will affect the activity of cathepsin B in the cell interior. Separate abstracts submitted by our lab are focused on the a1, a3, and a4 isoform knockouts.

Support or Funding Information

Denison University Anderson Summer Research Assistantship
Interaction between hyperbaric oxygen therapy and prostaglandin synthesis inhibition in the formalin pain test
Authors: Cruz-Martinez Sheila Belen , del Carmen Castillo-Hernández María , Guevara-Balcázar Gustavo , Correa-Basurto José , Ríos-Ramírez Diego Alejandro , Valencia-Hernández Ignacio
Published: April 2020

Background

Pain is the main symptom of inflammatory diseases, such as diabetes mellitus, hypertension, osteoarthritis among others. Lipopolysacaccharide from E. coli (LPS) has been used to induce an inflammatory process. Prostaglandins has a role in inflammation so that NSAIDs are useful pharmacological tools to treat it. Hyperbaric oxygen therapy (HBOT) has also been used in some of these diseases. However, information about pharmacological interaction with HBOT and its mechanism is scarce. So, the purpose of this work was to study the interaction between LPS, HBOT and NSAIDs using a formalin pain test.

Methods

96 adults male Wistar rats (150 to 200 g) were used and 2 groups treated with LPS or its vehicle were formed and each of them was divided in the next subgroups (n=8 each): Control, indomethacin, rofecoxib, HBOT, HBOT/indomethacin and HBOT/rofecoxib. 20 μL of 2% formalin was injected into the plantar surface of the right hind paw. Then nociceptive behavior was evaluated as the number of flinches in 1 min every 5 for 60 min. LPS and HBOT were applied 4 and 2.5 h prior to formalin respectively. Additionally, PGE2 and PGI2 was quantified using RP‐HPLC‐UV.

Results

Formalin produces a nociceptive behavior that decreased in the first 10 min (phase I) and in the next 50 increased to a plateau and then become to reduce (phase II, inflammatory). LPS increased and HBOT and drugs treatment reduced the phase II, while PGI2 levels were increased with LPS and reduced with HBOT and drugs. The HBOT seems to increase the indomethacin antinociceptive effect but HBOT effect was lesser than control and levels of prostaglandins were similar than in absence of HBOT. LPS treatment reduced the rofecoxib and HBOT effect increasing PGE2 and PGI2 levels respectively. Finally, when HBOT is associated with LPS and indomethacin, an increase on AUC of formalin and PGI2 levels was observed.

Conclusions

The HBOT and prostaglandin synthesis inhibition were effective antinociceptive interventions. However, indomethacin but not rofecoxib increased the HBOT antinoceptive effect diminishing PGI2 levels. In presence of an inflammatory process, the effect of HBOT and rofecoxib but no indomethacin, the antinociception was lost due to an increase in prostaglandin synthesis. Therefore, there are an interaction between HBOT and PG synthesis inhibition and the presence of inflammation reduced the beneficial effects of physical or pharmacological interventions.

Support or Funding Information

The authors would like to acknowledge CONACYT (557096) for their financial support
Strategies for Autonomic Nervous System Mapping of Fiber Composition and Neural Circuitry Using Transmission Electron Microscopy in Support of SPARC Projects
Authors: Biscola Natalia P. , Bartmeyer Petra M. , Zhang Nianhui , Havton Leif A.
Published: April 2020

The optimization of neuromodulation strategies is important for a development and refinement of therapies. The design of customized tools, such as electrical stimulating devices, depends on the detailed knowledge of nerve fiber composition and inter‐relationships between target organs, nerve populations, and synaptic circuitry. The autonomic nervous system gives support to organs and are composed by myelinated and unmyelinated fibers as well as pre‐ and post‐ganglionic neurons. Mapping an autonomic nerve such as the pelvic or vagus nerve that innervates peripheral tissues and understanding the synaptology of neurons in the cardiac and stellate ganglia may provide advanced knowledge for improved neuromodulation strategies. Transmission electron microscopy (TEM) is a high‐level technology that helps to identify and characterize axonal myelination and ultrastructure of unmyelinated fibers. TEM also allows for fine structural analysis of neural circuitry and innervation of autonomic targets. Immunogold detection techniques using TEM permits the identification and characterization of neuronal tracers in the nervous system. With the support from the Stimulating Peripheral Activity to Relieve Conditions (SPARC) program at NIH, we developed customized ultrastructural studies of the autonomic nervous system to facilitate refinement of neuromodulation strategies related to the autonomic control of cardiac, gastrointestinal, and lower urinary tract systems. Morphometric analysis is performed by automatic segmentation, and the development of a new strategy to calculate measurements of axon diameter, fiber diameter, myelin thickness, and G‐ratio provides a better understanding of fiber size for modelling studies. Both males and females from different species, including humans, are analyzed and compared. Implementation of common sampling techniques for nerves and ganglia across species provides increased rigor and reproducibility. TEM studies promote an improved understanding of functional studies and provide critical ultrastructural information for the development of new neuromodulation strategies.

Support or Funding Information

SPARC/NIH 1OT2OD026585‐01 and Dr Miriam and Sheldon G. Adelson Medical Research Foundation (AMRF)
Is the scapula a good indicator of taxonomy?: A geometric morphometric analysis using macaque monkeys
Authors: Kenyon-Flatt Brittany , von Cramon-Taubadel Noreen
Published: April 2020

In primates, scapular shape is often thought to correlate to locomotor patterns with distinct morphologies observed in arboreal versus terrestrial species. However, the extent to which intra‐generic morphological differences might be related to taxonomic differences as opposed to convergent locomotor behaviors remains to be tested. Thus, we tested the hypothesis that closely related, but behaviorally distinct, species may exhibit variation in their scapular morphology that match known phylogenetic relationships among the specious macaque monkey. Our sample (n=230) represented nine species; M. fascicularis, M. mulatta, M. fuscata, M. arctoides, M. radiata, M. nigra, M. nemestrina, and M. sylvanus and a Colobine outgroup (Trachypithecus cristatus). 3D scans of the left scapula were taken using an HDI structured light scanner and 5 landmarks and 61 sliding semilandmarks were applied to the scans to capture overall form. Following Procrustes superimposition, Canonical Variates Analysis (CVA) was performed and Procrustes coordinates were extracted and used for a Discriminant Function Analysis (DFA). The results of the CVA showed clear differences between Trachypithecus and macaques, as well as between M. fuscata and M. radiata and all other macaques. In the cross‐validated DFA, only 34.1% of individuals were correctly classified. The outgroup Trachypithecus showed a correct classification rate of 70%, as did M. nigra consistent with relatively distinct morphology in this taxon. Overall, patterns of morphological variation observed in the CVA do not match phylogenetic history, therefore suggesting environmental factors might influence the pattern of morphological variation among macaque species in novel ways, though this remains to be tested.

Support or Funding Information

This research is supported by a National Science Foundation Grant (NSF BCS‐1830745), Field Museum Visiting Researcher Fellowship, Primate Research Institute Cooperative Research Grant, University at Buffalo Asian Studies Grant, and the Mark Diamond Research Fund (SP‐18‐13).
Determining the Impact of Increased Physical Activity on Improving Sleep Quality in Young Adults
Authors: Darling Ashley , Chiu Alex , Henderson Eric , Autler Aaron S. , Weggen Jennifer B. , Decker Kevin P. , Garten Ryan S.
Published: April 2020

Disturbed sleep, defined as any alteration to normal sleep patterns, has been linked to poor cardiovascular health and an increase in cardiovascular disease (CVD) risk. These negative sleep patterns are highly prevalent with 35% to 41% of individuals in the United States reported some form of disturbed sleep. Although high amounts of physical activity (PA) are often associated with high sleep quality, little is known about PA’s effectiveness to improve different aspects of sleep (e.g. duration vs quality) and the mechanisms to which it can improve sleep quality.

Purpose

The study sought to determine the ability of increased PA to improve sleep efficiency in healthy young adults.

Methods

Nineteen young adults (25±4 yrs) were recruited for this study. Subjects wore an accelerometer (Actigraph GT3x‐BT) for a total of three weeks to record daily physical activity (step count; low, moderate, and vigorous physical activity) and sleep variables (efficiency, wake after sleep onset, number of nightly awakenings, time per awakening, and total sleep time). Subjects maintained normal physical activity levels for the first week (BL), then increased their step count by an average of 5,000 steps/day across the next two weeks (W1 and W2). Heart rate variability (HRV) and venous blood draws were collected weekly to assess sympathetic activity and inflammation, respectively.

Results

The physical activity intervention resulted in significant increases (p < 0.001) in step‐count
for both W1 (13163 ± 3184) and W2 (12168 ± 3619) when compared to BL (8648 ± 2615 steps/day). No significant differences from BL were observed when examining sleep efficiency (BL: 83.8 ± 6.4; W1: 85.5 ± 4.0; W2: 84.2 ± 6.1 %), sympathetic‐vagal balance, and inflammatory marker concentrations in W1 and W2. A significant correlation was revealed when assessing the change in sleep efficiency from BL to W1 (r = 0.81, p < 0.001) and BL to W2 (r = 0.52, p = 0.02) when compared to initial sleep efficiency values.

Conclusion

This study revealed that although young healthy individuals appear to lack improvements in sleep efficiency with an increase in physical activity, those who reported the lowest sleep quality had the greatest improvements in sleep efficiency following an increase in physical activity. Therefore, the findings of the study suggest that although increasing physical activity can improve sleep quality, a potential “ceiling effect” may occur, as when sleep quality is adequate, augmenting physical activity no longer has a substantial effect.
Investigation of G protein‐coupled receptor kinase 2 (GRK2) regulation of D2 dopamine receptor signaling: effects of GRK2 functional domains and pharmacologic and genetic inhibition of GRK2 activity
Authors: Schardien Kayla Anne , Soto Marta Sanchez , Inbody Laura , Berchiche Yamina , Kehrl John H. , Bradfield Clinton , Free R. Benjamin , Sibley David R.
Published: April 2020

The D2 dopamine (DA) receptor (D2R) signals through two major pathways, namely activation of G proteins and recruitment of β‐arrestin. Agonist binding stabilizes a receptor state that activates G proteins and facilitates receptor phosphorylation by one or more members of the G protein‐coupled receptor kinase (GRK) family, and this phosphorylation is known to enhance β‐arrestin recruitment and initiate the process of internalization for many GPCRs. Previously, we found that only GRK2 and GRK3 were involved in D2R phosphorylation in response to agonist stimulation (Namkung et al., JBC: 284: 15038, 2009). In this study, we further investigated the influence of GRK2 on receptor signaling, β‐arrestin recruitment, and internalization of the D2R. Overexpression of GRK2 was found to increase DA‐stimulated β‐arrestin recruitment and receptor internalization, whereas it suppressed D2R‐mediated inhibition of cAMP accumulation. In contrast, a catalytically inactive GRK2 mutant (GRK2‐K220R) did not affect any of these functional outputs suggesting that GRK2 kinase activity is required to influence D2R signaling. A disabling mutation of the GRK2 RGS homology (RH) domain (GRK2‐D110A) behaved similarly as WT GRK2, suggesting that this domain is not required for GRK2’s regulatory effects on the D2R. Finally, overexpression of a GRK2 construct containing a mutation in the pleckstrin homology (PH) domain (GRK2‐R587Q), which negates GRK2 interaction with Gβγ subunits, increased DA‐stimulated β‐arrestin recruitment and receptor internalization (more than observed with WT GRK2), but did not affect D2R regulation of cAMP accumulation, indicating that this GRK2 domain is involved in regulating some, but not all, aspects of D2R signaling. To study D2R regulation in the complete absence of GRK2, we utilized a cell line in which GRK2 expression was knocked out using CRISPR‐Cas9 technology. DA‐stimulated β‐arrestin recruitment and receptor internalization were decreased, but not eliminated, in the GRK2 KO cells suggesting that, while GRK2 may be facilitatory, it is not essential for these processes. In parallel studies we characterized the effects of Compound 101, a dual GRK2/3 inhibitor. Compound 101 was found to negate GRK2 interactions with the D2R and attenuate, but not eliminate, β‐arrestin recruitment and receptor internalization. In contrast, in the GRK2 KO cells, Compound 101 had no effect on the residual DA‐stimulated β‐arrestin recruitment. In conclusion, our data suggest that different GRK2 domains affect specific regulatory pathways of the D2R and that both agonist‐induced β‐arrestin recruitment and receptor internalization can occur in the absence of GRK2 interactions with the D2R.

Support or Funding Information

NINDS Intramural Research Program
Melatonin Prevents 1A Phase Ventricular Fibrillation in Porcine Acute Myocardial Infarction Model
Authors: Azarov Jan , Bernikova Olesya , Ovechkin Alexey , Mikhaleva Natalia , Khramova Daria , Tsvetkova Alena
Published: April 2020

Introduction

Ventricular fibrillation (VF) is a frequent complication of acute myocardial ischemia. Its prevention necessitates development of an antiarrhythmic cardioprotective strategy, and melatonin is considered to be a candidate antiarrhythmic agent. Though mechanisms of its action are largely unclear, generation of reactive oxygen species are thought to be its probable target. The objective of the present study was to find out an electrophysiological parameter subject to modification by melatonin and associated with VF incidence.

Methods

Acute ischemia was induced in 25 anesthetized pigs by a 40‐min ligation of the left anterior descending coronary artery. After tightening the ligature, 12 animals were given melatonin (4 mg/kg, i.v.) and 13 placebo (control group). Intramyocardial unipolar electrograms were led from flexible electrodes (a total of 48 leads) positioned across ventricular walls and interventricular septum (IVS). Local activation times (AT, dV/dt min during QRS), end of repolarization times (RT, dV/dt max during T wave) and activation‐repolarization intervals (ARI, difference between RT and AT) were measured. After experiments, the hearts were excised, and parameters of oxidative stress were determined in the ischemic and nonischemic zones of ventricular myocardium.

Results

In the control group, VFs were typically clustered during occlusion in 1A (1–5 min, 4 episodes) and 1B (17–40 min, 4 episodes) phases. Melatonin prevents development of 1A VFs (0 episodes, p=0.016) but did not modify occurence of 1B VFs (4 episodes). In the base of IVS (border ischemic zone), ATs in the period corresponding to 1A phase arrhythmias were shorter in the melatonin group as compared to the control group (Mean ± SD: 22±11 ms vs 39±18 ms, p=0.026, respectively) and were associated with VF incidence (OR 1.05 95% CI 1.01–1.10, p=0.023), while parameters of ventricular repolarization and oxidative stress did not differ between the treated and untreated animals.

In conclusion, melatonin treatment improves conduction in the border ischemic zone and thereby prevents early ischemic VFs independently of oxidative stress development.

Support or Funding Information

The study was supported by Russian Science Foundation (RSF 18‐15‐00309).