bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypotensive Effects of Ganaxolone are Associated with an Upregulation of GABAA Receptor Subunit Expression in Male Hypertensive Schlager Mice
Authors: Gueguen Cindy , Le-Pham Phuong-Nhan , Jackson Kristy L. , Paterson Madeleine R. , Esler Murray , Marques Francine Z. , Lambert Elisabeth , Head Geoffrey A.
Published: April 2020

Ganaxolone is a synthetic form of allopregnanolone, an allosteric modulator of gamma aminobutyric acid type A receptors (GABAAR). Allopregnanolone reduced blood pressure (BP) in male BPH/2J mice with neurogenic hypertension (Stevenson et al., 2017), which suggests that a lack of tonic inhibition in the forebrain mediates the hypertension. The hypotensive effect of allopregnanolone was associated with an upregulation of the GABAAR a4 and d subunit mRNA in the hypothalamus and medial amygdala of BPH/2J mice. The objective of this study was to investigate the temporal association of the antihypertensive effects of ganaxolone and the alteration in the different GABAAR subunits in the forebrain of BPH/2J mice.

12‐week‐old hypertensive BPH/2J and normotensive BPN/3J male mice were implanted with a telemetry probe to record BP and heart rate. Cardiovascular responses to stress tests and pentolinium were measured before and after administering vehicle or ganaxolone (5 mg/kg/day, subcutaneous minipumps) for 2 weeks. The mRNA expression of the GABAAR subunits was measured by real‐time PCR 1 to 2 weeks after the beginning of the treatment in vehicle or ganaxolone‐treated BPH/2J mice.

After 2 weeks, ganaxolone reduced BP by 10.7 mmHg (P<0.001) in male BPH/2J mice and reduced the pressor responses during restraint (−8.2 mmHg, P<0.001) and dirty‐cage switch (−12.4 mmHg, P<0.001) stresses compared with pre‐treatment. One‐week treatment with ganaxolone or vehicle had no effect on cardiovascular parameters on either strain. Ganaxolone‐treated BPH/2J mice had increased mRNA expression of α4‐subunit and showed a trend towards increased δ‐subunit of hypothalamic GABAAR compared to vehicle‐treated BPH/2J. None of these effects were seen at 1‐week post‐treatment and in BPN/3J mice.

In conclusion, ganaxolone reduced hypertension and the cardiovascular response to stress in male BPH/2J mice but only when upregulated a4 and d subunit mRNA expression of the GABAAR was observed. Therefore, it is likely that the main mechanism of the delayed antihypertensive effects of ganaxolone in neurogenic hypertensive mice is dependent on upregulation of specific GABAAR subunits.
Discovery of small molecules that sensitize Salmonella to polymyxin antibiotics
Authors: Thielen Michaela K. , Carlson Erin E. , May John F.
Published: April 2020

Salmonella enterica, a major bacterial cause of food‐borne illness, can survive in harsh environments due to its ability to sense and respond to toxic conditions. Salmonella senses a threatening environment, such as antibacterial conditions produced by the body’s immune system, and then activates the expression of genes necessary to survive within that environment. These genes are activated through the use of two component systems. Two component systems consist of an inner membrane‐bound histidine kinase and a response regulator protein located in the cytoplasm of the cell. The PhoP/PhoQ two component system is critical for the virulence of Salmonella and for Salmonella to resist antibiotics such as polymyxin B. Therefore, we tested commercially available small molecules capable of inhibiting the sensory histidine kinase to see if they were capable of sensitizing Salmonella to polymyxin B. Indeed, a few small molecules were successful at sensitizing Salmonella to polymyxin B. To determine how these molecules decrease polymyxin B resistance within Salmonella, we carried out several mechanistic studies. First, we tested whether these small molecules influenced Salmonella’s outer membrane permeability. This was tested by combining small molecules with vancomycin. Vancomycin is an antibiotic used effectively on gram‐positive bacteria that only contain a single membrane, but gram‐negative bacteria are intrinsically resistant to vancomycin due to their outer membrane. We found that vancomycin did not impact Salmonella’s resistance to polymyxin B. These results suggest that the small molecules do not affect outer membrane permeability. We also tested whether these molecules affect expression of PhoP‐activated genes. These molecules have been seen to decrease expression of a PhoP activated gene when transcriptional reporters were used. Using reverse transcription quantitative polymerase chain reaction (RT‐qPCR), we found that the small molecules decreased expression of some, but not all, PhoP‐activated genes. Overall, our results demonstrate that small molecules can target Salmonella’s ability to regulate gene expression and resist toxic conditions. This discovery represents a promising new way to develop antibiotic drugs to treat bacterial infections.

Support or Funding Information

This research was supported by the following funding: UWL Undergraduate Research and Creativity Grants (MKT), UWL College of Science and Health Dean’s Distinguished Fellowship (MKT), Research Corporation for Science Advancement, Cottrell Teacher Scholar Ambassadors for PUI – R1 Partnerships Award (EEC and JFM)
Effects of an acute 3‐h swimming exercise on insulin secretion capacity and its regulatory proteins of pancreatic islets in mice
Authors: Nonaka Yudai , Takeda Reo , Kano Yutaka , Hoshino Daisuke
Published: April 2020

Blood glucose homeostasis is regulated by glucose uptake in several peripheral tissues and insulin secretion from pancreatic β‐cells. While it has been well known that exercise improves glucose homeostasis by increased glucose uptake capacity of skeletal muscles, it is not fully elucidated whether acute exercise can change insulin secretion capacity in pancreatic β‐cells. Insulin secretion is regulated by several factors, such as autophagy, glucose transporter 2 (GLUT‐2) protein and phosphorylated Ca2+/calmodulin‐dependent protein kinase II (CaMKII). Exercise may affect these factors, because acute endurance exercise can activate autophagy activity in mouse pancreas. Therefore, this study was conducted to examine the effects of acute exercise on the capacity for insulin secretion and its underlying regulatory mechanisms. Male C57BL/6J mice were divided into two groups: a sedentary (CON; n=10) group or a single bout of 3‐h swimming exercise (EX; n=10) group. After the swimming exercise, oral glucose tolerance test (OGTT) and glucose‐stimulated insulin secretion in isolated islets were performed. The area under the curve (AUC) of blood glucose and insulin in the OGTT was not significantly different between the two groups. Although basal insulin secretion (2.8 mM glucose) in isolated islets did not differ between the two groups, insulin secretion in response to high‐glucose (16.7 mM) was enhanced in EX group compared with the CON group (+59%, p < 0.05). GLUT‐2 protein content but not autophagy‐related proteins and phosphorylated CaMKII protein in islets was higher in the EX group than in the CON group (+95%, p =0.05). These results suggest that an acute 3‐h period of swimming increased the insulin secretion capacity of pancreatic islets. The mechanism at least partially responsible for the favorable effects of exercise appears to be increased GLUT‐2 content in β cells.
Downregulation of Phosphoserine aminotransferase 1 promotes tumorigenesis in Glioblastoma
Authors: Chan Yung-chieh , Huang Shang-Pen , Chang Yu-Chan , Hsiao Michael
Published: April 2020

Glioblastoma (GBM) is a typical brain tumor with survival below ten percent at 5 years after diagnosis. The genetic alternation and histological features are integrated into the classification of gliomas for better clinical management of GBM. However, the development of therapeutic strategies and diagnosis for GBM patients remains poor. In this study, we found that downregulation of phosphoserine aminotransferase (PSAT1) mediated tumorigenesis of GBM. Analyses of available clinical datasets showed that PSAT1 downregulation was associated with poor survival of GBM patients. In western‐blot analysis of our GBM cell panel, PSAT1 protein was shown to be overexpressed in U87 and SNB75 cells, but downregulated in T98G and G9t cells. We overexpressed PSAT1 in T98G and G9T cells and knocked down PSAT1 in U87 and SNB75 cells for further study. The colony formation assay showed that PSAT1 overexpression inhibited clonal expansion in T98G and G9T cells. PSAT1 knockdown promoted cell growth in U87 and SNB75 cells. Furthermore, PSAT1 knocked‐down cells revealed significant tumorigenesis in mouse xenograft tumor model. The microarray assay indicated that downregulation of PSAT1 plays an important role in GBM tumorigenesis.
Mitochondrial Dysfunction Promotes Diabetes via A Previously Unrecognized Mechanism: Protein Succinylation
Authors: Lee Sooyeon , Annes Justin P.
Published: April 2020

Mitochondrial dysfunction is a central contributor to the development of β‐cell failure and Type 2 Diabetes (T2D). Succinate dehydrogenase subunit B (SDHB) is a key component of the Succinate Dehydrogenase (SDH) protein complex that strongly impacts mitochondrial function via 1) oxidation of succinate to fumarate in the TCA cycle and 2) electron transfer in the electron transport chain as Complex II. Loss of SDH activity has been observed in diabetic β‐cells (humans and rodents); however, the relevance of this observation to diabetes pathogenesis remains poorly understood. Consistent with prior work, we found reduced SDHB expression in the pancreatic islets of human diabetics. To investigate the links between mitochondrial dysfunction, β‐cell failure and diabetes, we generated mice with a β‐cell‐specific knockout of SDHB (SDHBβKO). We found that SDHBβKO mice develop early onset diabetes. At 5 weeks, prediabetic SDHBβKO mice demonstrate mildly elevated glucose levels with low insulin levels and normal insulin sensitivity. These in vivo findings were confirmed by a reduction in glucose‐stimulated insulin secretion in SDHBβKO islets. Furthermore, glucose‐induced β‐cell replication was impaired in SDHBβKO mice; consistent with impaired adaptive β‐cell expansion. Given the prominent role of glucose metabolism in controlling β‐cell insulin secretion and replication, we assessed the mitochondrial bioenergetics by Seahorse Assay and found that basal and glucose‐stimulated respiration were significantly reduced in SDHBβKO islets. Notably, loss of SDH is expected to increase succinate accumulation and promote protein succinylation, a recently described bulky post‐translational modification. Indeed, SDHBβKO islets demonstrated a dramatic increase in succinate levels (16‐fold) and protein succinylation (6‐fold), providing a potential mechanism for global mitochondrial dysfunction. Accordingly, expression of the desuccinylation enzyme, Sirt5, was increased in SDHBβKO islets. Metabolomics by mass spectrometry and transcriptomics by high‐throughput RNA sequencing revealed significantly altered mitochondrial metabolism. Finally, protein succinylation levels were increased in islets of diabetic db/db mice indicating lysine‐succinylation may be characteristic of diabetes. Taken together, these results demonstrate a surprisingly necessary role for the SDH complex in β‐cell function and introduce a pathogenic mechanism of diabetes based upon β‐cell metabolic dysfunction.

Support or Funding Information

Stanford Diabetes Research Center (SRDC) Pilot & Feasibility Grants Program (P30DK116074), Endocrinology Training grant (T32DK007217) and Child Health Research Institute at Stanford (UL1TR001085)
The Effects of Alcohol on MAP‐Kinase Tight Junction Regulation in Lymphatic Endothelial Cells
Authors: Herrera Matthew J. , Souza-Smith Flavia
Published: April 2020

Alcohol is known to cause lymphatic vessel hyperpermeability, leading to disrupted immune crosstalk between the gut and mesenteric lymph nodes. However, the mechanism of this alcohol‐induced hyperpermeability is unknown. The permeability of endothelial cell layers is correlated to decreased expression of certain proteins located at cell‐to‐cell contacts, known as tight junction (TJ) and adherens junctions (AJ). Proteins in these junctions, which predominantly include Claudin, Occludin, ZO‐1, and VE‐Cadherin, are shown to be regulated by MAP‐kinase proteins Erk1/2 and p38. In various endothelial cell types, alcohol increases phosphorylation of MAP‐Kinase proteins, leading to alterations in expression of TJ proteins. We hypothesize that alcohol induces lymphatic endothelial cell permeability via disruption of lymphatic endothelial cell TJs through activation of the MAP‐kinase pathway. To characterize the effects of alcohol on cellular permeability, we used and in vitro model of rat lymphatic endothelial cells. Alcohol‐supplemented medium was added at concentrations of 25 mM and 50 mM to confluent cells, and the transendothelial resistance across the cell monolayer, which is inversely related to permeability, was measured. After characterization of permeability, we measured phosphorylated states of MAP‐kinase proteins, specifically Erk1/2 and p38, at the time point that was associated with the maximum change in resistance. Our results show that alcohol increases the permeability of the endothelial cell monolayer compared to controls lacking alcohol. We also found that, at 50 mM alcohol, there was an increase in phosphorylated levels of p38 compared to controls. We predict that p38 might be affecting the expression or translocation of TJ and AJ proteins involved in forming and maintaining cell‐to‐cell adhesion, leading to increased permeability and lymphatic vessel leakage.

Support or Funding Information

LSUHSC Department of Physiology NIAAA ‐ K01 AA026640‐01 (FSS)
Impact of artificial sweeteners on cellular growth and signaling pathways
Authors: Parmar Nitika , Cuxil Lily Apen , Ruano Patricio
Published: April 2020

Artificial sweeteners such as saccharin, acesulfame, aspartame, neotame, and sucralose are found in a wide variety of foods today, particularly in zero‐calorie sugar substitutes (Equal, Sweet’ N Low, Splenda). Research conducted on animals and human epidemiological studies involving use of these compounds have provided contradictory results, in some cases indicating the compounds as safe and in others, as causing cancer. In our project, we sought to analyze the impact of six different synthetic sugar substitutes (found in Whole Earth, Equal, Splenda, Sweet’ N Low, Nutrasweet and Nutame) on bacterial and mammalian cell growth. The commercial substitutes as well as the purified ingredients found in these substitutes (erythritol, xylitol, stevia, monkfruit extract) were both tested. In this two‐phase project, E. coli cells were grown on minimal media lacking glucose but supplemented with the substitutes to determine the impact on growth. Bacterial cells were exposed to 0.4% of the compounds (0.4% is the standard glucose concentration found in rich LB media) and grown on media plates as well as in liquid cultures. Positive controls included minimal media plates containing glucose as well as rich media LB plates; negative controls included minimal media plates lacking any sugars. Our results indicate that bacteria are able to metabolize the sweeteners at different capacities but are unable to grow in acelsulfame‐K as well as the Whole Earth sweetener. A dose‐dependent profile to monitor whether bacteria will be able to utilize these compounds at higher doses (>0.4%) or will be able to survive if exposed to much lower doses (<0.1%) indicated that the growth increased with increasing concentration of the sweetener until 1%. In the second phase of our study we are growing mammalian normal (HEK293) and cancer cell lines in the presence of these substitutes at different concentrations and analyzing the impact specifically on the the mTOR signaling pathway that has been linked to a wide variety of cancers. Since some sugar substitutes have been linked to cancers in animals (e.g. sucralose to bladder cancer in rats) and considering the enormous use of these sweeteners today, this study will provide useful findings that could have important implications for their continued usage.

Support or Funding Information

This work is being supported by a UNIV498 Faculty‐Student Collaborative Research Award from CSU, Channel Islands to Nitika Parmar
Developing and Evaluating Recombinant Immunotoxins against Latent Epstein Barr Virus Infected Cells
Authors: Zhu Yuyi , Weldon John
Published: April 2020

Epstein Barr virus (EBV), commonly associated with acute infectious mononucleosis, is an oncogenic virus implicated in thousands of cancer‐related deaths every year worldwide. Estimates suggest that 90–95% of the population worldwide are infected with EBV. Individuals with healthy immune system can manage acute infections with minimal symptoms, but EBV often persists in a latent state even after the acute infection has cleared. Studies have shown a definitive link between latent EBV infections and the development of certain cancers such as nasopharyngeal cancer and Burkitt’s lymphoma, yet there are currently no targeted treatments against latent EBV induced cancers. A potential treatment for both latent EBV infections and EBV‐induced malignancies is recombinant immunotoxins (RITs) targeted to the virus‐specific membrane protein, LMP1. RITs are fusion proteins comprised of an antibody and protein toxin designed to identify and kill specific populations of cells. RITs using the bacterial toxin Pseudomonas exotoxin A (PE) have been extensively studied. Several RITs are in clinical trials, and one (moxetumomab pasudotox) has received FDA approval for the treatment of Hairy Cell Leukemia. Using a previously developed anti‐LMP1 antibody, we have cloned, expressed, and purified two anti‐LMP1 single‐chain variable fragment antibody joined recombinantly to either PE24 or PE38. We have begun to develop a HEK293 cell line that expresses LMP1 in order to evaluate the cytotoxicity of our anti‐LMP1 RITs. Subsequent experiments will evaluate these RITs as a therapeutic against EBV‐infected cell lines.

Support or Funding Information

This project was support by the Towson University’s Graduate Student Association and the Fisher College of Science and Mathematics
Mesenchymal Stem Cells Elicit Macrophages into M2 Phenotype via Improving TFEB‐mediated Autophagy to Alleviate Diabetic Nephropathy
Authors: Yuan Yujia , Zhu Lingling , Lu Yanrong
Published: April 2020

Diabetic nephropathy (DN) is a leading cause of end‐stage renal disease. Chronic inflammation is recognized as a key causal factor in the development and progression of DN, and the imbalance of M1/M2 macrophages (Mϕ) contributes to this process. Mesenchymal stem cells (MSCs) have been reported to prevent renal injuries via immune regulation in diabetic models, but whether these benefits are owing to the regulation of Mϕ, and the underlying signaling pathways are unknown. Here, we showed that MSCs elicited Mϕ into M2 phenotype and prevented renal injuries in DN mice, but these effects were abolished when the Mϕ were depleted by clodronate liposomes (Lipo‐Clod), suggesting that Mϕ were necessary for renal protection of MSCs in DN mice. Moreover, the MSCs promoted M2 polarization was attributable to the activation of TFEB and subsequent restore of lysosomal function and autophagy activity in Mϕ. Furthermore, in vivo adoptive transfer of Mϕin vivo (Mϕ from DN+MSCs mice) or MϕMSCs (Mϕ co‐cultured with MSCs in vitro) to DN mice improved renal function. While, TFEB knockdown in Mϕ significantly abolished the protective role of MϕMSCs. Altogether, these findings revealed that MSCs suppress inflammatory response and alleviate renal injuries in DN mice via TFEB dependent Mϕ switch.

Support or Funding Information

This work was supported by the National Natural Science Foundation of China (No. 81900666).
Comparisons of Nociceptive Behavior and NMDA receptor‐Mediated Synaptic Transmission between Mice Lacking D‐Serine Production and Degradation Enzymes
Authors: Kato Eiko , Saotome Toshiya , Yamaguchi Shigeki , Kansaku Kenji , Hori Yuuichi
Published: April 2020

D‐serine is the endogenous co‐agonist for the glycine‐binding site of the N‐methyl‐D‐aspartate (NMDA) glutamate receptor. D‐serine is produced by the enzyme serine racemase (SR) and is degraded by the enzyme D‐amino acid oxidase (DAO). Co‐activation of spinal NMDA receptors by D‐serine is known to play a significant role in the etiological mechanism of neuropathic and inflammatory pain. The present study aims to obtain further information on how D‐serine and its related enzymes (SR and DAO) are involved in the modulation of persistent pain.

The experiments were performed on both SR knock‐out (SR‐KO) mice and the DAO‐mutant mice that lack the enzymatic activity of DAO. We have previously shown that SR‐KO mice have less than half of the D‐serine content in the spinal cord and that DAO‐mutant mice have a significantly elevated level of D‐serine.The DAO‐mutant and SR‐KO mice used in this study are kindly provided by R. Konno (International University of Health and Welfare, Tochigi, Japan).

First, the nociceptive behavioral responses to formalin that is subcutaneously injected into a hind paw were analyzed. Results showed that the responses in the second phase, which are known be associated with NMDA receptor activation, were augmented both in SR‐KO mice and in DAO‐mutant mice.

Second, tight‐seal whole‐cell recordings were made from neurons in the superficial dorsal horn (SDH) in the lumbar spinal slices that were prepared from SR‐KO mice or from DAO‐mutant mice. Non‐NMDA and NMDA receptor‐mediated excitatory postsynaptic currents (non‐NMDAEPSCs and NMDA‐EPSCs, respectively) were pharmacologically isolated. Results show that the ratio of the amplitudes of NMDA‐EPSCs to that of non‐NMDA‐EPSCs was significantly smaller in SR‐KO mice and larger in DAO‐mutant mice compared with each corresponding control group and that the time constant of decay time course of NMDA‐EPSCs was significantly longer in SR‐KO mice.

Finally, quantitative real‐time RT‐PCR experiments were performed on RNA extracted from the SDH tissue. The results showed that the subunit composition of NMDA receptors altered in SR‐KO mice and DAO‐mutant mice. The most significant observation is that the expression level of the NR2B subunit of NMDA receptors increased in the SR‐KO mice.

These observations might indicate that D‐serine and its related enzymes (SR and DAO) influence the activation level of NMDA receptors and their subunit composition and thus affect nociceptive synaptic transmission in the SDH of the spinal cord and influence nociceptive behavior.

Support or Funding Information

This work was supported by JSPS KAKENHI Grant Number JP18K16462 (EK).
Exploration of the antidepressant‐like effect of repeated administration of Nigella fixed oil in rats
Authors: Kadil Youness , Tabyoui Imane , Badre Latifa , Tahiri Nadia Jouti , Filali Houda
Published: April 2020

Due to the confirmation of the therapeutic properties of oils extracted from Nigella, and the constraints associated with antidepressant prescriptions, the reliance on phototherapy seems to be beneficial. The objective of the present paper is to investigate the behavioral effect of Nigella fixed oil, as well as exploring the histopathological impact on the structure of the hippocampus after exposure to the Unpredictable Chronic Mild Stress procedure in rats.

Animals were randomly divided into five groups of eight. The stress procedure used in this study combines many stressful situations. These situations were randomly assigned with a frequency of two stressors per day throughout the 5‐weeks. Behavioral evaluation of antidepressant activity was based on the forced swimming test and histological evaluation of the hippocampus of rats with optical microscopy.

Treatment by nigella and fluoxetine significantly reduces the struggling time during the test day of the forced swim test. Histopathological analysis showed that control treatments result in more loosely arranged cells, significant apoptotic neurons characterized by an irregular appearance, and pyknotic hyperchromatic. As well as a reduction in the thickness of the pyramid layer compared to the groups treated with nigella and fluoxetine.

The results demonstrated the effect of Nigella in the forced swimming test, as well as the improvement of the pyramidal cell organization and neuronal degeneration. These data seem to be consistent with other studies, in which the neuroprotective properties of nigella refer to thymoquinone.
Characterization of the Troglitazone Induced Apoptosis in a Human Colon Cancer Cell Line
Authors: Przybelski Samantha , Ulloa Massiel E. , Rudie Melisa , Cornett Megan , Strom David
Published: April 2020

Troglitazone (TRO), a peroxisome proliferator‐activated receptor (PPAR) agonist, was a drug used as an anti‐inflammatory and anti‐diabetic agent. PPARɣ specifically is a known member of the steroid receptor superfamily and is a ligand‐activated nuclear transcription factor that regulates lipid and glucose homeostasis. As a PPARɣ activator, TRO, a thiazolidinedione, has been shown to have the effect of inducing cell differentiation but also inhibiting cell growth, proliferation, and angiogenesis thereby facilitating apoptosis, making it a potential candidate to study as a cancer chemotherapeutic agent.

Through the use of flow cytometry, TRO‐induced apoptosis of tissue culture grown colorectal cancer cells was examined. Apoptosis was quantified by analyzing cells that contain less than the 2N DNA content (one hallmark of apoptosis). A dose response curve was determined for increasing concentrations of TRO vs sub2N DNA content. These studies confirm that TRO was able to induce apoptosis in our cell lines at concentrations greater than 30mM.

Next we examined which PPAR receptors subtypes are being activated by TRO. HCT116 cells were exposed to various concentrations of TRO for 24hr then RNA was isolated from these cells. Specific gene targets to each PPAR receptor subtypes were identified and using RT‐PCR, changes in mRNAs expression was examined. Targets for PPARdelta and PPAR gamma were consistently upregulated. We are working to refine these experiments to get better determinations on which receptors are activated by TRO.

These experiments confirm that TRO induces apoptosis in HCT116 cells. Additionally, we have identified changes in at least one target of PPAR receptors in response to TRO exposure. Further experiments are needed to exactly determine which PPAR receptors are being activated by TRO, and whether the apoptosis medicated by TRO is a result of PPAR receptor activation.
Optimization of Inhibitory Peptide Delivery to Block GPCR Induced Inflammation
Authors: Antoniades William , Grimsey Neil
Published: April 2020

A key mediator of angiogenesis and inflammatory response in chronic lung disease is the mitogen‐activated kinase (MAP), p38α. The typical activation pathway for p38α uses MAP kinase kinase 3 (MKK3), and MKK6 to phosphorylate and activate p38α. However, an atypical p38α signaling pathway was discovered in a family of pro‐inflammatory G‐protein coupled receptors (GPCRs). The atypical pathway uses transforming growth factor β activated kinase 1 binding protein‐1 (TAB1) as opposed to MKK3/6 to interact with p38α. The binding of TAB1 to p38α induces the autophosphorylation of p38α which leads to increased proangiogenic signaling. Recent studies have highlighted the use of cell‐penetrating inhibitors that specifically block atypical p38 signaling, leaving MKK3/6 dependent signaling intact to maintain normal cellular function. We have shown that we can label peptide inhibitors with a red fluorescent protein which can be transiently expressed in endothelial cells. Using a lentivirus we are generating endothelial cell lines stably expressing an array of RFP linked inhibitor peptides targeted for exosomal packaging. Purified peptide loaded exosomes/nanocarriers are then used to deliver inhibitors to vascular cells in vitro. Our current studies focus on assessing the cellular uptake of peptide loaded exosomes by observing the mRFP using confocal microscopy and immunoblotting. We predict that exosomally delivered peptides inhibit p38 signaling by GPCR ligands in endothelial cells. These studies will demonstrate the potential for exosomal nanocarriers to deliver peptide inhibitors to block GPCR inflammation.
Functional Characterization of X Antigen Genes (XAGEs)
Authors: Terrazas Sari , Ramanathan Saumya
Published: April 2020

Cancer‐testis antigens (CTAs) are a group of genes that are normally expressed in the testis but aberrantly expressed in many different types of cancers. X‐linked antigens (XAGEs) are CTAs that are located on the X chromosome and that remain understudied and their role in cancer remains unclear. The specific expression of XAGEs in cancer cells make them a potential target for cancer therapy. The goal of this research project is to elucidate the biochemical function of XAGE genes and their protein products in normal and diseased states. We will first generate an atlas of cancer cell lines that display XAGE expression. Then we will perform gain and loss of function studies to determine what pathways are perturbed in the presence and absence of XAGEs. We will also determine binding partners of these proteins by IP‐Mass Spectrometry. In summary, our studies will shed light on the biochemical functions of the enigmatic XAGE proteins.

Support or Funding Information

HRD1764201
Sex‐dependent Effects of High‐fat Diet and Stress on CNS Inflammation and Ceramides Production
Authors: Sambolin-Escobales Lubriel , Tirado-Castro Lizmarie , Fonseca-Ferrer Wilfred , Santos-Aviles Kimberly , Pacheco-Cruz Dariangelly , Colon Maria , Hernandez Anixa , Chompre Gladys , Porter James
Published: April 2020

The intake of a high‐fat diet (HFD) and exposition to stress are associated with development of a neuroinflammatory state in the central nervous system (CNS). Previously we found high levels of long‐chain (C18:0 and C20:0) ceramides in blood of rats fed an HFD that correlated with increased immobility in the forced swim test, suggesting that ceramides could contribute to the development of depressive‐like behavior. In the current study, we aimed to determine whether adding short‐term unpredictable stress (STUS) would exacerbate the effects of HFD on neuroinflammation and ceramide metabolism in the ventral hippocampus (VH) and the development of depressive‐like behavior. We hypothesized that exposure to HFD and STUS would cause a neuroinflammatory state and increased levels of long‐chained ceramides in the VH leading to depressive‐like behavior. To test this, male and female Sprague Dawley rats were administered either a low‐fat or high‐fat diet for eight weeks, while other groups of female and male rats were administered a low‐fat or high‐fat diet for 10 weeks with the inclusion of 4 days of random unpredictable stressors. After sacrifice, we assessed the expression of microglial (Iba‐1), neuroinflammatory state (NLRP3, HMGB1 and TNF‐α) and ceramide production markers (CerS5, CerS6, SMPD1 and SMPD3) in the VH. Female HFD rats showed increased expression of TNF‐α and HMGB‐1 independent of STUS, while male rats increased expression of Iba‐1, TNF‐α and NLRP3 after exposition to STUS, suggesting a sex‐dependent activation of inflammatory mediators. The assessment of enzymes that produce long‐chained ceramides revealed increased CerS5, CerS6 and SMPD1 mRNA only in the VH of female HFD rats exposed to STUS, suggesting sex differences in the production of long‐chained ceramides due to combined HFD and STUS. VH tissue and blood were collected to quantify ceramides by gas chromatography‐mass spectrometry (GC‐MS). Based on the expression of the enzymes involved in the production of ceramides, we expect that the GC‐MS assay will show increased C16 ceramides in female rats exposed to HFD and STUS. After diet completion, rats were subjected to sucrose grooming test and forced swim test to assess anhedonia and behavioral despair. Female HFD with STUS showed trending increases in grooming activity, reduced immobility and increased swimming time while male rats did not present any behavioral changes, suggesting less depressive‐like behavior in female rats. In conclusion, an HFD in combination with STUS causes sex‐dependent changes inflammatory state and long‐chained ceramides production without increasing depressive‐like behavior.

Support or Funding Information

NIH‐NIGMS R25GM082406 (PHSU RISE Graduate Training Program), U54 MD007579, NIGMS/INBRE P20 GM103475, and NIH‐NIGMS R25GM096955 (UPR‐Ponce PRISE Program).
Pulmonary Toxicity of Nine Sand Dusts Generated at Hydraulic Fracturing Sites in Comparison to Respirable Crystalline Silica
Authors: Fedan Jeffrey S. , Barger Mark , Leonard Stephen S. , Thompson Janet A. , Snawder John E. , Dozier Alan , Coyle Jayme , Kashon Michael , Roberts Jenny R.
Published: April 2020

The levels of respirable crystalline silica (RCS) at gas wells employing hydraulic fracturing (fracking) sites have been reported to exceed regulatory standards. Inhalation of RCS in work places may cause silicosis and other diseases in workers. While the effects of inhalation of RCS in humans and animal models has been well‐characterized, the pulmonary hazards to worker associated with RCS inhaled at fracking sites has not been investigated. In this study, nine fracking sand dusts(FSDs) collected by vacuum dust controls from different fracking sites in the US were compared to MIN‐U‐SIL 5® (MIN) with respect to particle size, mineral composition, Si radicals and pulmonary toxicity. Chemical analysis revealed that total mineral content of the FSDs other than Si ranged widely from 9.9 g/kg to 32.5 g/kg when compared to 1.4 g/kg for MIN. This heterogeneity was confirmed by energy‐dispersive X‐ray spectroscopy (EDS). Hydrodynamic particle diameter of neat FSDs using dynamic light scattering (DLS) indicated non‐concordant, mono‐ orbi‐modal spectral distributions of particle sizes; the spectrum for MIN was mono‐modal. Electron paramagnetic resonance (EPR) analysis indicated the presence of the Si radical in MIN but not in neat FSDs. After incubating the FSDs in water the Si radical was revealed in some samples, indicating occlusion of the radical in neat FSDs. To examine pulmonary toxicity, rats were intratracheally instilled with a single dose of FSD or MIN (159 or 500 μg/rat), or vehicle. Thirty days later, bronchoalveolar lavage (BAL) was performed to assess the development of pulmonary inflammatory responses: changes in lactate dehydrogenase (LDH) and differential cell counts. This time point has been shown previously to be one at which pre‐silicotic responses to MIN are observed. As expected, MIN elicited a dose‐dependent increase in LDH levels, total BAL cells, and total alveolar macrophages, polymorphonuclear lymphocytes and eosinophils. As a group, the FSDs did not elicit these responses. These findings indicate that, in many respects, the chemical and biophysical characteristics, and the biological effects of the FSDs and MIN after intratracheal instillation, have distinct differences.

Support or Funding Information

NIOSH 6927ZLDC
Role of Na+/H+ Exchanger 1 in Regulating Resistance to Apoptosis in Pulmonary Arterial Smooth Muscle Cells
Authors: Munson Micheal , Yun Xin , Jiang Haiyang , Philip Nicolas , Huetsch John , Shimoda Larissa
Published: April 2020

Vascular remodeling within the pulmonary arterioles is a key component of pulmonary arterial hypertension (PAH). In a rat model of severe PAH, the SU5416‐Hypoxia (SuHx) model, pulmonary arterial smooth muscle cells (PASMCs) demonstrate resistance to apoptosis and contribute to vascular remodeling. Na+/H+ exchange (NHE) is increased in SuHx PASMCs; however, few studies have elucidated the functional role of NHE in these cells. Previous studies have shown a link between NHE and the expression of CHOP, a critical mediator of apoptosis. Therefore, we tested the hypothesis that altering NHE1 expression and or activity is sufficient to modulate apoptosis in PASMCs. PASMCs were isolated from distal pulmonary arteries dissected from vehicle‐treated, male, Wistar rats or rats exposed to hypoxia after an injection of SU5416 (SuHx rats). PASMCs were treated with vehicle, a combination of a pharmacologic inhibitor of NHE (EIPA) and hydrogen peroxide (H2O2), an apoptosis‐inducing agent, or either EIPA or H2O2 alone. Apoptosis was measured via Hoechst staining. In other experiments, control PASMCs were infected with a viral construct to overexpress wild‐type NHE1 (AdNHE1), a translocation‐disabled NHE1 mutant (AdE266I), or green fluorescent protein (AdGFP) as a control. NHE activity was measured via the ammonium‐pulse technique. SuHx PASMCs exhibited decreased CHOP expression and minimal apoptosis rates in response to H2O2 when compared to control PASMCs. EIPA had no effect on unstimulated apoptosis in control cells, but induced apoptosis in SuHx PASMCs at baseline and sensitized the cells to H2O2. AdNHE1, but not AdE266I, increased NHE1 activity. Interestingly, both AdNHE1 and AdE266I conferred a resistance to H2O2‐induced apoptosis when compared to the AdGFP‐infected cells. Overall, these findings suggest that increased NHE1 may be responsible for attenuating apoptotic responses in SuHx PASMCs and that increasing NHE1 levels in normal PASMCs is sufficient to promote increased survival via an ion‐translocation‐independent function.

Support or Funding Information

Supported by NIH grants HL073859, HL126514 and HL084762
Role of Mucin‐1 in Carcinomas
Authors: Link Tina , Caruso Olivia , Kona Abhishek , Lu Allison , Saberi Zara , Shabazaz Tara , Vijay Varun
Published: April 2020

Mucin 1 (MUC1) is a large, heavy O‐glycosylated mucin glycoprotein with a molecular weight of 250 to 500 kDa, depending on the level of glycosylation, and extends 200–500 nm from the cell surface. MUC1 spans the cell membrane at the apical surface of normal simple epithelia and the surfaces of some hematopoietic cells. MUC1 is comprised of three major domains: the cytoplasmic tail, transmembrane, and extracellular domains. The cytoplasmic tail (CT) domain is highly conserved across species and interacts with various cell signaling networks. The transmembrane domain is composed of hydrophobic amino acids to anchor MUC1 in the membrane. The extracellular domain is not strictly conserved, but always consists of a series of 20–100 tandem repeats enriched in serine, threonine and proline residues providing sites for the heavy O‐linked glycosylation characteristic of mucin‐type glycoproteins. The extracellular domain contributes MUC1’s function as an inhibitor of cell‐cell and cell‐extracellular matrix adhesion. The heavy glycosylation of MUC1 normally serves to protect cells from infection and also lubricates the cell surface. In normal biology, MUC1 serves as an inhibitor of embryo attachment to the uterine cell surface and controls early aspects of the reproductive process. In cancer, carcinomas often overexpress MUC1 due to a combination of gene duplication events as well as stimulation of MUC gene expression by cytokines produced by cells of the immune system in their efforts to destroy the cancer cells. Cancer cells also lose their normally polarized distribution of MUC1 and instead express the glycoprotein over their entire surface, MUC1 protects tumor cells from the immune system in two ways. First, by inhibiting the cell contact required for cell‐mediated immunity, e.g. by NK cells. In addition MUC1 expressed at tumor cell surfaces or shed by the action of cell surface proteases is immunosuppressive, presumably by interacting with immune checkpoint inhibitors. Various mouse models have been used to conduct research about MUC1, showing the connection between MUC1 overexpression in mouse mammary glands and cancer development and other processes. MUC1 serves as a commonly used serum marker for various cancers and is being explored as a target for certain therapeutic approaches. The Walton SMART team has designed a 3D model of MUC1 to investigate the relationship between its structure and function.

Support or Funding Information

MSOE Center for Biomolecular Modeling
Concomitant NFE2L2 and CTNNB1 mutations in a subset of HCC patients: Synergy between Nrf2 and Wnt pathway in hepatocarcinogenesis
Authors: Tao Junyan , Liu Silvia , Bell Aaron , Monga Satdarshan P
Published: April 2020

Background

Hepatocellular cancer (HCC) remains a major clinical need with gradually increasing incidence and currently lacks precision therapy. Thus, there is a need to better characterize molecular drivers in HCC and generate relevant models to test biology and therapies. In the USA, CTNNB1 mutations occur in 26% of all HCCs, however they alone are insufficient to lead to tumors as shown by preclinical studies suggesting cooperation with other pathways.

Methods

We assessed TCGA and other publically available databases for aberrations that co‐occur with CTNNB1 in HCC cases. We used sleeping beauty transposon/transposase (SBT) to express such clinically relevant mutants in a subset of hepatocytes in vivo in mice using hydrodynamic tail vein injection (HTVI). Specifically, we co‐delivered T41A‐CTNNB1 and wild type (WT)‐NFE2L2, G31A‐NFE2L2 or T80K‐NFE2L2 into 6–8 weeks old FVB mice via SBT‐HTVI and assessed tumorigenesis over time. WGS was performed on mouse tumors to identify differentially expressed genes and pathways. Similarity between the mouse model and human HCC subset was addressed by overlap between the most significantly altered pathways.

Results

Of the 377 HCC patients in TCGA, 99 patients showed CTNNB1 mutations. Analysis for Nrf2 pathway showed 15 HCCs with NFE2L2 and 18 with KEAP1 mutations. 12 patients showed coexistence of CTNNB1 mutations with wither NFE2L2 (n=6) or KEAP1 (n=6). Co‐expression of T41A‐CTNNB1 with G31A‐NFE2L2 or T80K‐NFE2L2 but not WT‐NFE2L2 led to HCC by 13–14 weeks. H&E staining confirmed the presence of well‐differentiated HCC. Immunostaining and WB showed HCCs to be strongly positive for b‐catenin targets (GS and pmTOR) and also for Nrf2 targets including NAD(P)H dehydrogenase [quinone] 1 and peroxiredoxin‐1. Next RNA‐Seq analysis on tumor‐bearing livers in mice using FDR=0.1 as cutoff showed many significantly altered pathways. Analysis of Nrf2‐CTNNB1 mutant HCC in TCGA also identified 64 pathways that were significantly altered using the FDR=0.1 as cutoff. 21 common and significant pathways were identified between our mouse model and patients, which have well known correlation with HCC development.

Conclusions

Thus, Nrf2 and Wnt pathway cooperate in HCC pathogenesis in a subset of patients. Co‐expression of mutant NFE2L2 and mutant CTNNB1 leads to HCC in mice with notable similarity in pathway alterations to the human HCC subset. This will be a valuable model to study biology and treatment for this subset of HCC cases.

Support or Funding Information

This work was supported by NIH grants R01CA204586 to S.P.M
The sigma‐1 agonist PRE‐084 protects against energy depletion induced barrier disruption and also enhances glycolysis in HUVECs
Authors: Motawe Zeinab Y. , Farsaei Forouzandeh , Abdelmaboud Salma S. , Cuevas Javier , Breslin Jerome W.
Published: April 2020

The sigma‐1 receptor (σ1) is a well‐known endoplasmic reticulum (ER) chaperone that resides at the mitochondria‐associated ER membrane, having an important role in calcium signaling between ER and mitochondria. Expression of σ1 is present in all human tissues with specific predominance in arteries, liver, fibroblasts and brain. Our laboratory has shown that σ1 activation in lymphatic vessels elicits nitric oxide‐dependent lymphatic relaxation, a finding that suggested the potential important role of σ1 in endothelium. Other recent work has highlighted that σ1 may contribute to healthy endothelial barrier function, although little is known about the mechanisms involved. We hypothesized that σ1 helps protect the endothelial barrier disruption by stabilizing cellular bioenergetics. Cultured human umbilical vein endothelial cell (HUVEC) monolayers were used to model the endothelial barrier. Electric cell‐substrate impedance sensing (ECIS) was used to study the contribution of σ1 to endothelial barrier function. Western blotting was used to quantify σ1 protein expression. Endothelial bioenergetics were determined with extracellular flux analysis using glycolytic rate and ATP rate assays. The endothelial‐specific contribution of σ1 was assessed using with the σ1‐selective agonist PRE‐084 and by specific siRNA knockdown of σ1. The mitochondrial un‐coupler Carbonyl cyanide m‐chlorophenyl hydrazine (CCCP) was used to model mitochondrial dysfunction in HUVEC. Our results show that activation of σ1 with PRE‐084 50 μM, 100 μM, 150 μM, 200 μM significantly enhances barrier function of HUVEC monolayers in a concentration‐dependent manner. Knockdown of σ1 with siRNA reduces baseline barrier function and diminishes the ability of PRE‐084 to enhance TER. The decrease in TER elicited by CCCP (10 μM) is significantly amplified in cells with siRNA‐mediated knockdown of σ1 expression, and is reversed by PRE‐084 (100 μM) in cells with normal σ1 expression. HUVEC treated with PRE‐084 display higher levels of glycolysis and glycolytic ATP production than control cells. This response to PRE‐084 was not evident in the presence of σ1 knockdown. Collectively, our results suggest that σ1 plays an important role in maintenance of baseline endothelial barrier function, and that activation of σ1 can enhance barrier function. Moreover, σ1 activation also can rescue endothelial barrier dysfunction caused by disruption of mitochondrial ATP production, likely due to its ability enhance glycolysis. Thus, σ1 may represent a future therapeutic target to rescue endothelial barrier dysfunction.

Support or Funding Information

This work was supported by NIH Grant R01GM120774.