bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chemical Biology Approaches for Genome‐Wide Screening of GPCR Trafficking
Authors: Lobingier Braden , Tsvetanova Nikoleta , von Zastrow Mark 
Published: April 2020

G protein‐coupled receptor (GPCRs) are the largest family of membrane‐bound signal transduction molecules. Many of these receptors have a complex lifecycle which includes residence at various intracellular organelles: endoplasmic reticulum, Golgi, plasma membrane, endosomes, and lysosomes. Movement of the receptor between these organelles is essential for GPCR function and mis‐trafficking can alter, blunt, or entirely disrupt GPCR signaling. Given the complexity of GPCR trafficking, we sought to develop chemical biology methods which would allow forward genetic screens to be performed in mammalian cells. Here we describe a novel approach, based on a genetically encoded peroxidase called APEX, allowing for quantitative fluorescent detection of GPCR expression. The fluorogenic substrate used in this method is membrane permeable while the fluorescent oxidation product is stably deposited into cells, allowing for subsequent single‐cell analysis and sorting. We demonstrate that APEX is protease‐labile and its activity is rapidly quenched upon trafficking of the GPCR‐APEX transgene to the lysosome. Specific chemical or genetic perturbation of GPCR trafficking to lysosomes preserves the enzymatic activity of APEX, which opens the door to forward genetic analysis. We combine this chemical biology method with genome‐wide CRISPRi screening to identify genes involved in the trafficking of the delta opioid receptor from ER (synthesis) to lysosome (degradation). Together with our previous work using APEX for high‐resolution proximity labeling, we present a comprehensive pipeline for proteomic and genomic characterization of GPCR cell biology.Support or Funding InformationNIH DA043607
Optimization of Cell‐Free Protein Synthesis Through Identification of Rate‐Limiting Factors
Authors: Smith Philip E. J. , Burrington Logan R. , Levine Max Z. , Watts Katharine R. , Oza Javin P. 
Published: April 2020

We have implemented the Genetic Code kit into classrooms at Cal Poly, San Luis Obispo for a hands‐on inquiry‐based module of transcription and translation. Our Genetic Code kit is based on cell‐free protein synthesis (CFPS) which captures the transcription and translation machinery required for protein synthesis in vitro, allowing for direct manipulation of protein production. By obviating the use of a living cell, CFPS allows early‐career STEM students to observe protein synthesis. To observe the process, superfolder green fluorescent protein (sfGFP), is used as a reporter protein. The detectable fluorescence of sfGFP gives students real‐time feedback of protein synthesis occurring. However, this process takes upwards of three hours for complete synthesis and maturation. To be more applicable in an educational setting, CFPS needs to occur in a more time‐efficient manner. Thus, this work focuses on identifying the rate‐limiting factors of CFPS. These rate‐limiting factors can be organized into two classes. The first class consists of energy sources and small molecules, and the second class consists of protein‐based translation factors. Direct titrations of these small molecules and proteins into CFPS reactions were performed. Here, we report our findings of key additives that increase the translation rate in cell‐free. Additionally, miscellaneous small molecules, such as osmolytes and detergents, have also been found to increase the reaction rate. The translation factors currently being studied are: prokaryotic initiation factors, elongation factors, release factors, ribosome recycling factor and the methionyl transferase. By optimizing the process of CFPS, the platform can become time‐efficient for a single lab setting. To date we have decreased reaction time by 30%, significantly improving the broad applicability of the Genetic Code Kit in classrooms.Support or Funding InformationOur research is funded by the Center for Applications in Biotechnology, the Bill and Linda Frost Fund, and NSF‐1708919.
Role of the Renal Nerves in Regulating SGLT2 inhibitor‐induced Diuresis and Natriuresis in rats with Heart Failure
Authors: Katsurada Kenichi , Nandi Shyam S. , Sharma Neeru M. , Patel Kaushik P. 
Published: April 2020

Sodium‐glucose cotransporter 2 (SGLT2) inhibitors promoting glucose excretion are widely used to treat type 2 diabetes. Recently, the clinical trial in both diabetic and non‐diabetic heart failure (HF) patients with reduced ejection fraction demonstrated that SGLT2 inhibitor prevents worsening HF. However, the underlying mechanisms have not been fully elucidated. The present study was conducted to assess whether the effects of SGLT2 inhibitor on urine flow, sodium and glucose excretions are changed in HF. To address the underlying mechanisms the mRNA and protein levels of renal SGLT2 are investigated. HF was induced in rats by left coronary artery ligation. Further, to assess the contribution of renal nerves in the regulation of renal SGLT2, four weeks after ligation surgery, surgical bilateral renal denervation (RDN) was performed. Acute experiments were performed one week after RDN (five weeks after coronary ligation). Urine flow, sodium and glucose excretion responses to SGLT2 inhibitor dapagliflozin (0.5 mg/kg, i.v.) were greater in HF than Sham (Urine flow 15.8 ± 1.8 vs. 8.3 ± 1.1 μl/min/gkw, Sodium excretion 2.23 ± 0.35 vs. 1.13 ± 0.18 μEq/min/gkw, Glucose excretion 0.84 ± 0.06 vs. 0.52 ± 0.08 mg/min/gkw at 120 min, P<0.05, n=5–7). RDN significantly attenuated these responses to dapagliflozin in HF (Urine flow 9.8 ± 1.2 vs. 15.8 ± 1.8 μl/min/gkw, Sodium excretion 1.31 ± 0.17 vs. 2.23 ± 0.35 μEq/min/gkw, Glucose excretion 0.66 ± 0.04 vs. 0.84 ± 0.06 mg/min/gkw at 120 min, P<0.05, n=5–7). Western blot analysis indicated that SGLT2 protein expressions in the renal cortex were higher in HF than Sham (SGLT2/GAPDH 0.83 ± 0.06 vs. 0.58 ± 0.05, P<0.05, n=6). RDN significantly reduced enhanced protein levels of SGLT2 in the renal cortex of rats with HF (SGLT2/GAPDH 0.60 ± 0.07 vs. 0.83 ± 0.06, P<0.05, n=6). In contrast, RT‐PCR analysis indicated that RDN increased mRNA levels of SGLT2 in the renal cortex of rats with HF (SGLT2/18sRNA 1.08 ± 0.28 vs. 0.23 ± 0.06, P<0.05, n=6), possibly a compensatory change to SGLT2 protein expressions. Immunohistochemistry of renal sections confirmed the enhanced staining for SGLT2 in HF. These findings suggest that increases in urine flow, sodium and glucose excretion responses to SGLT2 inhibitor are enhanced in HF due to the upregulation of renal SGLT2. Renal sympathoexcitation in HF might enhance the expression and functional activity of renal SGLT2, thereby contribute to subsequent pathophysiology of renal sodium and water retention associated with HF.Support or Funding InformationSupported by Japan Heart Foundation and NIH grants DK 114663 and HL62222.
GAL3 Regulates Smooth Muscle Cell Survival in PAH via its NWGR Motif
Authors: Haigh Stephen , Li Xueyi , Bordan Zsuzsanna , Chen Feng , Meadows Mary Louise , Stepp David , Barman Scott , Fulton David 
Published: April 2020

Pulmonary arterial hypertension (PAH) is a complex and fatal disorder characterized by an unrelenting increase in pulmonary arterial pressure. Excessive proliferation of pulmonary artery smooth muscle cells (PASMC) leads to increased vascular resistance and eventually right ventricular failure and death. Our laboratory has identified a key role for Galectin‐3 (GAL‐3 or Lgals3) in driving PASMC proliferation and elevated pulmonary pressures. However, the mechanisms by which GAL‐3 alters PASMC behavior remains poorly understood. GAL‐3 contains a BH1 like domain that has an NWGR sequence with high homology to the anti‐apoptotic protein, BCL2. Our central hypothesis is GAL‐3 upregulation plays an important role in driving PASMC proliferation through repression of apoptosis via the NWGR domain. In order to test the role of this domain we have made an adenoviral vector encoding the GAL‐3 G182A mutation and have shown that it expresses equally to a WT GAL‐3. We have also made a GAL‐3 KO rat and have isolated GAL‐3 PASMCs from these animals (RPASMC). Our in vitro studies show that overexpression of GAL‐3 G182A cause a decrease in viability using flow cytometry upon treatment overnight with 0.5% serum containing media(p<.001). However there was no significant difference in survival(p=.840) when cells were conditioned in normal media. Proliferation experiments similarly showed no significant difference between GAL‐3 WT and G182A in KO human aortic endothelial cells(HAEC) and human PASMC (p>.05). When exposed to apoptotic stimuli (10ng/mL TNFα and 1μg/ml Cycloheximide) GAL‐3 G182A overexpressing GAL‐3 KO RPASMCs had significantly (p<.001) decreased vitality as compared to GAL‐3 WT overexpressing cells. Overall our data suggests that the NWGR domain of GAL‐3 is important in regulating PASMC survivability, and our future work will investigate the role of GAL‐3 mediated apoptosis in animal models of PAH.Support or Funding InformationF31HL147428R01HL125926‐03
Angiotensin II‐Induced Increase in Blood Pressure in Mice Consuming High Salt and High Fructose Diet: Females Not Protected
Authors: Fan Liming , Vallejo Jessica , Sung Austin , Rouch Alexander 
Published: April 2020

Hypertension presents major risk factors for cardiovascular and renal disease. High salt and high fructose (HSHF) consumption contribute to hypertension via salt retention and obesity. Sex differences in blood pressure regulation are well known. Some studies have shown protection for females from angiotensin II (AngII)‐induced and dietary‐induced increase in blood pressure. Our results indicate that HSHF consumption increases blood pressure in male and female mice. Also, AngII administration increased blood pressure in male and female mice while consuming normal diet with water, but no sex differences were noted (Rouch, A. FASEB J. 32(1) A904.6, 2018). The objective of this study was to determine the effects of AngII on blood pressure, sodium excretion, and urine flow rate in male and female CD‐1 mice while consuming a HSHF diet. Four groups of mice included male‐vehicle, male‐AngII, female‐vehicle, and female‐AngII (n=6/group). Mice at four weeks of age were placed in metabolic cages for a 5‐day baseline period followed by a ten‐day AngII period where AngII was administered via an Alzet® osmotic minipump containing either saline (vehicle) or AngII to yield 1mg/kg/min. All mice consumed a diet of 4% salt with fluid containing 1% NaCl and 20% fructose ad libitum. Systolic blood pressure was measured daily via the tail‐cuff method (CODA, Kent Scientific). Excessive fluid retention evidenced by a significant increase in body weight of 4–5 g occurred in the AngII‐treated mice of both sexes in the first 5 days of the AngII period. One mouse of each AngII‐treated group died before the end of the AngII period while the other mice recovered to their normal body weight by the end of the ten‐day period. AngII increased systolic blood pressure (mmHg) in females from 108.0 ± 3.4 to 130.0 ± 6.1 (p<0.0001) and in males from 101.1 ± 1.3 to 127.5 ± 3.2 (p<0.0001). In both AngII‐treated groups, daily urine flow rate and sodium excretion demonstrated biphasic patterns of significant decreases in the first 5 days of the AngII period followed by increased levels to achieve fluid and sodium balance. We conclude that AngII‐infusion during HSHF consumption in mice presents with potentially fatal salt and water retention. No sex differences in the AngII‐induced increase in SBP or renal handling of sodium were measured.Support or Funding InformationOK‐INBRE SURP
Inhibiting Progesterone induced blocking factor induces markers of endothelial dysfunction and inflammation in pregnant rats
Authors: Amaral Lorena M. , Witcher Alexis C. , Comley Kyleigh , Cottrell Jesse N. , Cunningham Mark W. , Ibrahim Tarek 
Published: April 2020

Preeclampsia (PE) is a leading cause of maternal and perinatal morbidity in the U.S. While the pathogenesis remains unclear, PE is characterized by new onset hypertension associated with progesterone deficiency, elevated cytolytic natural killer cells (cNK), inflammation, and endothelial dysfunction. Progesterone is essential in the initiation and maintenance of pregnancy. We have shown that progesterone supplementation regulates endothelial function and suppresses endothelin‐1 (ET‐1) and stimulates nitric oxide (NO) in response to placental ischemia during pregnancy. In addition, progesterone signals the synthesis and release of progesterone induced blocking factor (PIBF) from lymphocytes in order to regulate the proinflammatory balance of early pregnancy. However the role of PIBF in PE pathology is not well examined. This study was designed to test the hypothesis that inhibition of PIBF causes inflammation and increases markers of endothelial dysfunction and hypertension during pregnancy. Rabbit anti‐PIBF IgG (0.25, low dose‐LD or 0.50 mg/mL, high dose‐HD) was administered intraperitoneal on gestation day (GD) 15 to normal pregnant Sprague Dawley (NP) rats, on GD 18 carotid catheters were inserted and on GD 19 blood pressure (MAP) and samples were collected. MAP in NP rats (n=7) was 99± 3 mmHg, which increased to 116± 2 in NP+ anti‐PIBF LD (n =10) and 113±4 mmHg in NP+ anti‐PIBF HD (n=6), p<0.05. Neither placental weight nor pup weight was affected by PIBF blockade. Plasma TNF‐alpha levels were 35±8 pg/mL in NP rats and increased to 84±21 pg/mL in NP+ Anti‐PIBF HD (n=4), p<0.05. Circulating total NK cells were 67± 11 in NP rats (n=4), which decreased to 36±4 in NP+ Anti‐PIBF HD however, cytolytic NK cells were 0.6 ± 0.2 in NP which were increased to 3.0±1 in NP+ Anti‐PIBF HD, p<0.05. Importantly, circulating NO levels were 44± 11 μM in NP rats (n=5), which significantly decreased to 21±1 μM in NP+ Anti‐PIBF (n=6) HD, p<0.05. Moreover, renal cortex PPET‐1 levels increased 15 fold in NP+ Anti‐PIBF (n=6) HD compared to NP rats (n=5). Collectively, our study demonstrates that inhibition of PIBF causes hypertension, increased TNF alpha while causing signs of endothelial dysfunction, all of which are associated with PE, thus indicating the importance of progesterone signaling pathways for the maintenance of endothelial function and inflammation during healthy pregnancy.Support or Funding InformationThis work was supported by National Institutes of Health grants RO1HD067541‐06, P20GM121334 and AHA 19CDA34670055
SPARC: Development of a TRPA1 reporter mouse model
Authors: Bahia Parmvir K. , Kim Seol-Hee , Harsányiová Jana , Kollarik Marian , Taylor-Clark Thomas 
Published: April 2020

TRPA1 is a non‐selective cation channel activated by electrophiles including environmental irritants, and pungent stimuli. It is expressed in a subset of nociceptive sensory neurons and thus plays a key role in defensive reflexes. The contribution of TRPA1 to certain physiological effects is complicated by its expression in non neuronal cell types such as fibroblasts and epithelial cells, so it is not clear to what extent TRPA1 effects are restricted to sensory afferents. We have created a mouse model that co‐expresses the red fluorescent protein tdTomato in TRPA1 expressing cells. A TRPA1‐Flp knock‐in strain was generated by incorporating a 2A‐FlpO after the last endogenous TRPA1 exon. The TRPA1‐Flp was crossed with a tdTomato reporter strain carrying a frt‐flanked STOP codon (RC::FLTG) to produce cells that produce tdTomato in TRPA1 expressing cells. From these animals, we collected sensory vagal and thoracic dorsal root ganglia to visualize tdTomato‐expressing cells in combination with immunohistochemistry. We also collected brain, brainstem, spinal cord and esophagus to visualize tdTomato‐expression to map their central and peripheral projections. tdTomato positive cells were observed primarily in the nodose ganglion and the majority of these coincided with TRPV1 expression. There was also a population of tdTomato expressing cells in the dorsal root ganglia. We confirmed, using calcium imaging, that these tdTomato positive cells are activated by the TRPA1 agonist AITC. We saw strong tdTomato expression in the nucleus of solitary tract (NTS) and in the lamina II region of the dorsal horn of the spinal cord but no expression was evident in the brain. Finally, we were able to observe tdTomato expression in peripheral fibers extending into different regions of the esophagus. In summary, we have created a transgenic knock‐in FlpO mouse model which selectively labels vagal sensory neurons with AITC‐sensitivity. This strain can be used to identify peripheral and central terminals of TRPA1 expressing nerves.Support or Funding InformationThis study is funded by National Institutes of Health Common Fund SPARC OT2 (2016–2019): “Functional mapping of peripheral and central circuits for airway protection and breathing”
Dietary Dependent Alteration of Blood‐Brain Barrier Tight Junctional Protein Expression
Authors: Reeb Chris , Sandoval Karin , Wooten Joshua , Harris Mathew , Schaller Megan , House Austin , Witt Ken A. 
Published: April 2020

The impact of diet on brain health extends to the blood‐brain barrier (BBB). Western diets high in saturated fatty acids, such as lard, increase the risk for neurodegenerative disease and cerebrovascular dysfunction. Whereas, fish‐oil (FO) diets containing long‐chain polyunsaturated fatty acids can form products that reduce inflammation and have been identified to have cerebrovascular benefit. However, the impact of dietary fats on the BBB endothelial cell tight junctions remains unclear. BBB tight junction integrity is governed by key proteins, including claudin‐5, occludin, and zonula occluden‐1 (ZO1). This study examined the effect of lard and FO based diets on these key tight junctional proteins.Male C57BL/6J mice received a diet containing either 10% lard, 10% FO, 41% lard or 41% FO over 32 weeks. Cortical brain tissue was harvested at conclusion of treatment, with protein (Western blot) and mRNA (RT‐qPCR) expression analyses performed. Body weights and blood chemistry measures were also evaluated. Two‐way ANOVAs performed, with Tukey post‐hoc test used to determine if means significantly differed, with significance set at P<0.05.No differences in mRNA expression were observed across diets. Neither the type or percentage of fat had any impact on occludin protein expression. ZO1 protein expression was significantly higher with FO compared to lard. A significant interaction was found between the type and percentage of fat on claudin‐5 protein expression. Respective to lard, claudin‐5 protein expression significantly decreased as the percentage of fat increased from 10 to 41%. Respective to FO, claudin‐5 protein expression significantly increased as the percentage of fat increased from 10 to 41%. Respective to 10% fat, claudin‐5 protein expression was significantly lower in FO compared to lard. Respective to 41% fat, claudin‐5 protein expression was significantly lower in lard compared to FO.Study identified ZO1 and Claudin‐5 protein expression changes within the brain are linked to diet. Data supports that diets high in FO have a greater expression of key tight junctional proteins, which suggests enhanced BBB integrity.Support or Funding InformationSupported by the NIH, National Institute of Neurological Disorders and Stroke (grant R21NS090282).
Resting Heart Rate Variability at Sea Level Predicts Arterial Desaturation during Acute Exercise in Hypoxia
Authors: Short Kelsey Jean , Roarty Margaret C. , Amaya Jessika , Kodali Sruthi , Nylund Zachary J. , Jackson Jaren T. , McKee Kellyn E. , Harris M. Brennan , Kambis Kenneth W. 
Published: April 2020

Predicting responses to acute hypoxia based on physiologic measures at sea level may be valuable in anticipating adverse responses to acute hypoxia. The purpose of this study was to examine the relationships between heart rate variability (HRV) and arterial oxygen desaturation in response to acute hypoxia exposure at rest and during exercise.METHODS13 subjects (6 male, 7 female) aged 20.6±0.3 years rested in a supine position for 10 min. Average resting HRV and arterial oxygen saturation (%SpO2) were calculated from the last 5 min. A VO2peak test to determine aerobic fitness (44.1±3.0 ml/kg/min) was performed on a stationary cycle at sea level. Subjects also performed a submaximal bike test for 10–15 min at 65% of HRmax achieved during the VO2peak test in normobaric hypoxia equivalent to an altitude of 3500 meters.RESULTSHRV (RMSSD 54.5±5.7) was significantly correlated (r=0.227, P<0.05) to the percent change (9.7±0.7) in %SpO2 from rest (97.4±0.2) to exercise (88.0±0.8) in normobaric hypoxia.CONCLUSIONSHRV at rest appears to be predictive of the degree of arterial desaturation during exercise in hypoxia equivalent to an altitude of 3500 meters. Current wearable technology that can measure HRV could be used to predict potential adverse responses to exercise in hypoxic conditions such as altitude.Support or Funding InformationThe Doug Morton/Marilyn Brown Endowment for Biomedical Research, The Foundation for Aging Studies and Exercise Science Research, and The Borgenicht Program.
Endothelin Receptor Antagonism Increases Cerebral Perfusion in Humans
Authors: Ward Aaron T. , Carter Katrina J. , Bolin Shawn E. , Eldridge Marlowe W. , Hagen Scott A. , Walker Benjamin J. , Lee Jeffrey W. , al-Subu Awni M. , Wieben Oliver , Schrage William G. 
Published: April 2020

Microvascular cerebral perfusion (CP) is critical for adequate oxygenation of neuronal tissue in the brain. However, the mechanisms underlying proper CP regulation in humans have yet to be fully defined. Endothelin is a potent endogenous vasoconstrictor and appears to have a modest role in blood flow regulation in many peripheral vascular beds in healthy individuals. We tested the hypothesis that endothelin receptor antagonism will not influence CP in healthy young adults. Six male subjects (age: 24±4 yrs; BMI: 23±2 kg/m2) participated in the single‐blind study in which endothelin receptor antagonist (Ambrisentan, 10 mg) or placebo was administered orally. Only males participated in the current study due to potential teratogenic effects of Ambrisentan. Resting CP was quantified via 3T MRI using pseudo‐continuous arterial spin labeling (ASL) normalized to gray matter volume approximately 120 minutes following dosing. Blood pressure, heart rate, and end‐tidal CO2 were monitored. Data were analyzed via paired t‐tests and reported as mean±SD. Significance was taken as p≤0.05. Heart rate (57±8 vs. 59±7 bpm), mean arterial pressure (81±7 vs. 79±8 mmHg) and end‐tidal CO2 (34±6 vs. 37±3 mmHg) was not different between placebo and Ambrisentan, respectively (all p>0.05). Compared to placebo, Ambrisentan increased cerebral perfusion (31±3 vs. 34±2 mL/100g/min; p<0.05), representing a 8±5% increase. Regionally, Ambrisentan demonstrated a tendency to increase CP in brain regions supplied by the anterior cerebral circulation (frontal lobe: 36±11 vs. 45±5 mL/100g/min, p=0.05; parietal lobe: 35±10 vs. 43±5 mL/100g/min, p=0.08; temporal lobe: 35±10 vs. 43±6 mL/100g/min, p=0.07; basal ganglia: 35±9 vs. 41±5, p=0.09; cingulate: 39±10 vs. 47±5 mL/100g/min, p=0.08). In contrast, CP in regions supplied by the posterior circulation did not increase to a similar extent (occipital lobe: 37±9 vs. 43±7 mL/100g/min, p=0.13; cerebellum 30±16 vs. 38±6 mL/100g/min, p=0.27). Contrary to our hypothesis, these data suggest endothelin contributes to resting CP by increasing microvascular tone, an effect that may be more pronounced in the anterior brain regions. These preliminary findings contribute to resolving potential mechanisms mediating normal CP in healthy individuals and form the basis of future studies exploring the influence of elevated endothelin signaling in conditions like diabetes or heart failure.Support or Funding InformationADA 1‐16‐ICTS‐099