bluuebunny/crossref_metadata_2025_split huggingface dataset:
SRSF3 and hnRNP K Antagonistically Regulate Splicing of Large Exons
Authors: Kawachi Toshihiko , Masuda Akio , Takeda Jun-ichi , Ito Mikako , Hamaguchi Tomonari , Ohno Kinji 
Published: April 2020

Mammalian internal exons are usually between 50 and 200 bp long. Their average length is ~150 bp, and only 5% of them are larger than 300 bp. Although previous studies showed that splice site recognition becomes less efficient with increasing exon size, the mechanism how large exons are correctly spliced is not well understood. To investigate the role of RNA binding proteins (RBPs) in the splicing of large exons, we analyzed 842 publicly available RNA‐seq datasets from GEO, in which 48 canonical splicing factors were individually knocked down. MISO analysis was performed to detect exons that were specifically included or excluded by knockdown of an RBP, and average lengths of these exons were calculated. Our analysis revealed that SRSF3 is the top RBP that promotes inclusion of the largest exon set (390 exons per analysis, 337 bp on average).Interestingly, most of the SRSF3‐dependent large exons are annotated as constitutive exons in the public database. Consistently, these exons have high splice site strengths. Motif analysis revealed that the SRSF3‐dependent large exons are greatly enriched with C‐rich sequences, which are preferentially recognized by SRSF3. In addition, our analysis also detected the enrichment of C‐stretches, which are potential binding sites for hnRNP K. ENCODE eCLIP shows that HNRNPK extensively binds to the SRSF3‐dependent large exons. To examine whether hnRNP K is involved in the regulation of SRSF3‐dependent splicing of large exons, we knocked down Hnrnpk along with Srsf3‐knockdown using C2C12 myoblasts. RNA‐seq analysis revealed that Srsf3‐silencing alone induced skipping of 374 exons, of which average length is 395 bp. Co‐silencing of Srsf3 and Hnrnpk rescued skipping of 142 exons by Srsf3‐silencing, of which average length is still long, 458 bp. These results indicate that substantial number of large exons require SRSF3 to prevent their skipping by hnRNP K.To investigate the molecules associated with SRSF3 and hnRNP K in the splicing of large exons, we next conducted proteomic approach that defines the native protein complexes in the chromatin fraction. Cells expressing FLAG tagged SRSF3 or hnRNP K were lysed under the physiological salt concentration, and isolated chromatin fractions were immunoprecipitated with At‐FLAG antibody. Mass spectrometry analysis of the immunoprecipitants revealed that SRSF3 is mainly associated with U1 snRNPs, U2AFs, and SF1, and to a lesser extent with hnRNPs, while hnRNP K is preferentially associated with hnRNPs and U1 snRNPs, but not with U2AF or SF1. Thus, SRSF3 and hnRNP K are involved in early but distinct stages of spliceosome assembly. Together, our analysis identified the antagonistic splicing regulation of SRSF3 and HNRNPK on C‐rich large exons.
Regulation of transient receptor potential canonical 4 activity by phospholipase C δ1
Authors: Kang Hana , Ko Juyeon , Myeong Jongyun , Kwak Misun , So Insuk 
Published: April 2020

Transient receptor potential canonical (TRPC)4 and TRPC5 channels are non‐selective calciumpermeable cation channels that maintained by phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2) and inactivated due to its depletion. Phospholipase C (PLC) is an enzyme that cleaves phospholipids and the PLCδ is the most calcium‐sensitive form among the isozymes that stimulated by physiological concentration of Ca2+. Here, we investigated the regulation mechanism of TRPC4 by the Ca2+‐PLCδ‐PI(4,5)P2 signaling cascade. In order to identify the interaction between ion channel and PLCδ protein, we implied co‐immunoprecipitation and fluorescence imaging including Föster Resonance Energy Transfer. We evaluated the activity of channels with electrophysiological recording in HEK293 cells expressing TRPC channels. Here, we demonstrate that TRPC4 directly interacts with PLCδ1. We also found that PLCδ isozymes are activated by the calcium through opened channels but not by the cytosolic calcium increase. Our study established the PLCδ1 inhibits overall TRPC4’s currents activity, but mainly regulates the basal TRPC4 currents to have a characteristic low basal activity. These regulation of PLCδ1 on TRPC4 activity occurs depend on PI(4,5)P2 hydrolysis. Resultantly, PLCδ1 is considered to be a negative regulator of TRPC4.Support or Funding InformationThis research was supported by the National Research Foundation of Korea, which is funded by the Ministry of Science, ICT (Information & Communication Technology), and Future Planning (MSIP) of the Korean government (2018R1A4A1023822 to I.S.S.). H.Kang, J.Ko., J.Myeong., and M.Kwak. were supported by the BK plus program from the MSIP.
Subcellular Location‐Dependent Activation and Signaling of the Delta‐Opioid Receptor
Authors: Crilly Stephanie E. , Weinberg Zara Y. , Ko Wooree , Puthenveedu Manojkumar A. 
Published: April 2020

G protein‐coupled receptor (GPCR) signaling from intracellular compartments is an emerging idea in the field. Classically, GPCRs have been thought to signal mainly from the plasma membrane. However, GPCRs localized to endosomes and the Golgi are a critical component of receptor signaling. GPCR location affects signaling at multiple levels, from generation of second messengers to transcriptional responses, and even physiological responses. One physiologically relevant receptor that shows location‐dependent signaling is the delta‐opioid receptor (DOR), an attractive alternative target for pain management. DOR is localized primarily to intracellular compartments, including the Golgi, in neuronal cells. Relocation of these receptors to the cell surface increases the ability of DOR agonists to relieve pain, suggesting that the subcellular location and signaling of this receptor are closely linked and important for receptor pharmacology. However, the mechanisms by which DOR relocation to the plasma membrane from Golgi membranes influences receptor pharmacology are poorly understood. Live cell imaging with nanobody and mini‐G protein biosensors indicates receptor localization influences ligand‐induced recruitment of active‐conformation biosensors. Additionally, we used cAMP and calcium sensors to study how DOR localization influences receptor signaling. Together, our data support a model in which DOR activation and signaling are spatially regulated. This spatial regulation may underlie the complexity of physiological effects associated with DOR activation and provide new ways to modulate receptor signaling.Support or Funding InformationM.A.P. was supported by NIHGM117425 and NSF1517776. S.E.C was supported by NSF Graduate Research Fellowship DGE1256260.
Unfolding the Mysteries of Oxygen Sensing ‐ A Comprehensive Analysis of the Hypoxic Response in Zebrafish Gills One Cell at a Time via Single Cell RNA Sequencing
Authors: Pan Wen , Godoy Rafael , Cook David , Scott Angela L. , Nurse Colin A. , Jonz Michael G. 
Published: April 2020

All vertebrates “sense” changes in O2 through specialized cells, called chemoreceptors, which interact with the peripheral and central nervous systems to maintain O2 homeostasis. Aquatic vertebrates are especially prone to fluctuations in environmental O2 availability. Therefore, it is vital for them to readily detect changes in PO2 and make appropriate respiratory and cardiovascular adjustments. Teleost fish, such as zebrafish (Danio rerio), have O2 chemoreceptors, called neuroepithelial cells (NECs), present in the gill epithelium and undergo membrane depolarization and vesicular recycling when exposed to acute hypoxia. NECs have also been shown to increase in number and size following chronic hypoxia. Here we present a comprehensive analysis of the effects of hypoxia on gill cells including NECs through a transcriptomic study. Using the transgenic line ETvmat2:GFP, zebrafish were acclimated to normoxia (PO2 = 160 mmHg) or severe hypoxia (PO2 = 35 mmHg ) and maintained for 14 days. Isolated cells from the distal ends of gill filaments, areas rich in NECs, were analyzed using single cell RNA sequencing (scRNA‐seq) analysis. Four normoxia (control) and four hypoxia‐treated biological samples were individually labelled before scRNA‐seq library construction using the 10X Genomics chromium technology. A total of 45,000 gill cells were consequently sequenced on a NextSeq500 (Illumina) sequencer with a read depth of 17,000 per cell. Our preliminary results separated 21 cell clusters and identified one cluster to be NECs based on gene expression. Highly differentially expressed genes in NECs included tph1a encoding tryptophan hydroxylase 1, the rate limiting enzyme in 5‐HT biosynthesis, and sv2 encoding synaptic vesicle 2 protein. Other differentially expressed genes in NECs are involved in mitochondrial oxidative metabolism and ATP synthesis. Moreover, differential expression analysis showed a clear shift in the transcriptome of NECs following 14 days of chronic hypoxia. NECs in the hypoxia group showed a high expression of genes involved in stress‐regulated polyubiquitination in contrast to the control group. The transcriptomic change implies a change in dynamics of the NEC population. We are the first group to conduct a single cell transcriptomic study in zebrafish gill cells. Our study is expected to provide a complete atlas for the hypoxic response in single gill cells and serve as a platform for future work on peripheral oxygen sensing.Support or Funding InformationThis research project is funded by Natural Sciences and Engineering Research Council of Canada (NSERC).
Investigating the Roles of Orexinergic System in Methamphetamine‐induced Addiction
Authors: Lee Chan , Bae Hui Jin , Jang Jung-Hee , Park Gyu Hwan 
Published: April 2020

Methamphetamine (METH) is a powerful neurotoxic psychostimulant characterized to affects activity of dopamine transpoters leading to blockage of dopamine (DA) uptake into the synaptic vesicle as well as DA reuptake through the plasma membrane inducing synaptic DA release, which resulted in continuous excess extracellular DA levels in the synaptic cleft. Orexinergic system has been reported to play crucial roles in regulation of arousal, wakefulness, and motivated behaviors for drug abuse. In this study, we have investigated the roles of orexin and/or orexin receptors on METH‐induced addictive behaviors by conducting conditioned place preference (CPP) test using SB334867, an orexin‐1 receptor antagonist. The changes in the dopaminergic system integrity and orexin‐related signaling molecules were examined to elucidate underlying molecular mechanisms. C57BL/6 mice were administered with METH (1 mg/kg, i.p.) alternately on each other day for 8 days, allowed to undergo withdrawal period without injection of METH for additional 8 days, and then challenged again with METH. METH‐administered mice exhibited increased levels of orexin and orexin‐1 receptor in the brain regions of hippocampus, striatum, hypothalamus, and amygdala. The application of SB334867 significantly attenuated the acquisition, expression, and reinstatement of the METH‐induced CPP. These findings suggest that orexinergic system may play a role in the acquisition and relapse of METH‐induced addictive behaviors, thereby providing a novel therapeutic target for the METH addiction.Support or Funding InformationThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT)
Attenuation of Aortic Remodeling with Low Doses of Neprilysin/Angiotensin II Receptor Blockers Sacubitril/Valsartan in a Fibrillin‐1‐Deficient Model of Marfan Syndrome
Authors: Bernatchez Pascal , Sauge Elodie , White Zoe 
Published: April 2020

IntroductionSacubitril is a neprilysin inhibitor indicated for heart failure patients with reduced ejection fraction (HFReF), which causes protective natriuretic peptide accumulation. To maximize efficacy and minimize side effects, sacubitril is administered in combination with valsartan, a blood pressure‐lowering angiotensin II receptor blocker (ARB). We and others have shown that ARBs are capable of blood pressure lowering‐independent effects on vascular and lung remodeling, but whether the sacubitril/ARB combination shows greater anti‐vascular and lung remodeling effects is unknown.ObjectiveSince ARBs are routinely prescribed to patients with Marfan syndrome, which is a fibrillin‐1 mutation‐induced connective tissue disease that causes aortic widening, dissection and lung emphysema, we sought to test the anti‐remodeling effects of low‐doses sacubitril/valsartan combination on the vascular and pulmonary complications associated with Marfan syndrome in mice.ResultsTreatment with low dose valsartan and sacubitril/valsartan (pure forms in drinking water) did not significantly lower blood pressure when compared with vehicle group in the C1039G fibrillin‐1 model of Marfan syndrome. There was a non‐significant inhibitory trend of Marfan‐associated aortic root widening with valsartan and especially sacubitril/valsartan, whereas only the latter induced a significant and near‐complete (74%) inhibition of Marfan‐associated ascending aorta widening (P<0.008). None of the treatment groups significantly reduced emphysema, loss of lung volume, change in hysteresis, tissue damping and resistance associated with Marfan syndrome.ConclusionSacubitril/valsartan combination may be more clinically relevant than valsartan alone in settings of aortic dilation, even at low doses.Support or Funding InformationSupported by Novartis Canada
Insulin Analog with Additional Disulfide Bond Forms a Structurally Secure, Thermodynamically Stable Molecule
Authors: Candler Noelle Riley , Horth Shelby Noelle 
Published: April 2020

Insulin is a hormone secreted by the pancreatic cells. In vivo, normal insulin contains 3 disulfide bonds that account for structural instability and low melting point. In this experiment, an insulin analog was attempted to be made mechanically and thermodynamically more stable than human insulin (HI). It was hypothesized that the addition of a fourth disulfide bond (4SS‐insulin) would be more stable as previous attempts at other types of transformational change to the A‐chain and B‐chain were unsuccessful. By adding a fourth disulfide bond in positions A10/B4, the outcome would be a significant clinical application for the treatment of insulin‐dependent diabetes as scientists are constantly trying to find insulin analogs that would potentiate the effects of blood sugar lowering. To experiment, the fourth disulfide bond was successfully formed using yeast as a template. They tested fragility (fibrillation), low pH, and thermodynamic stabilization, finding that the fourth disulfide bond made insulin more mechanically and thermodynamically stable. Once this was complete, in vivo experimentation on rates demonstrated the effect on blood glucose and in vitro experimentation on lipogenesis in rat adipocytes. In vivo, 4SS‐insulin had a longer and more profound effect on blood glucose than HI. In vitro, 4SS‐insulin had a greater affinity for insulin receptors than HI. The Kohler High School MAPS Team used 3‐D modeling and printing technology to examine the relationship between a structurally secure fourth disulfide bond and the thermodynamic stability of an insulin analog in comparison to typical HI. The clinical applicability of this new biomedically engineered 4SS‐insulin is significant. Patients could be able to self‐inject less insulin less frequently, helping with affordability and improved glycemic control. Additionally, the storage of insulin becomes more convenient as 4SS‐insulin can withstand a 34.6 °Celsius increase in melting temperature.PDB: 4EFX
Immuno‐Sympathoinhibitory targeted Electroacupuncture Treatment Enhanced Blood Pressure Lowering Responsiveness in Middle‐Aged Hypertensive Patients
Authors: Tjen-A-Looi Stephanie , Xie Lifang , Fu Liang-Wu , Nguyen Lan , Malik Shaista 
Published: April 2020

We have shown that 8‐week electroacupuncture (EA) at P5‐6 and St36‐37 reduces blood pressure (BP) (70% effectiveness, BP decrease of ≥ −6 mmHg in peak or average systolic or diastolic) and norepinephrine in a group of male and female (38–75 year) patients with mild to moderate hypertension (HTN). During the 8 EA sessions, the onset of the BP reduction was observed by week 4, while these patients were not on antihypertensive medications. BP reduction with 30‐min EA once a week treatment at P5‐6+St36‐37 as such is defined as sympathoinhibitory. Interestingly, only 59% of middle‐aged and elderly (46–75 years) patients with HTN are responsive to this sympathoinhibitory treatment. To this end, we formulated a targeted treatment that addresses low grade chronic inflammation present in hypertensive patients. High sensitivity C‐reactive protein (hs‐CRP) is a marker of systemic chronic low‐grade inflammation. We hypothesized that immuno‐sympathoinhibition by EA treatment decreases BP and hs‐CRP in majority of middle‐aged and elderly patients with HTN. Participants were not on antihypertensive medications and randomly allocated to either sympathoinhibitory (n=12) or immuno‐sympathoinhibitory (n=9) EA treatment. After completion of the 8‐week course of treatment, the BP lowering responsiveness (≥ −6 mmHg) to the immuno‐sympathoinhibitory EA was 88% (P=0.016) while the sympathoinhibitory was 58% (P=0.042). The hs‐CRP appeared to be reduced in immuno‐sympathoinhibitory EA treated patients (−6.7±3.1 %, n=5) following 8‐week EA treatment in contrast to patients treated with sympathoinhibitory EA (0.03±0.15%, n=12). Thus, EA targeted towards the immune and sympathetic systems combined may be important in lowering the BP and chronic low grade inflammation in majority of middle‐aged and elderly hypertensive patients.Support or Funding InformationAdolph Coors Foundation
Changes in Individual and Team Cognition in High Stress Extreme Underwater Saturation Environment Under Intense Workload
Authors: Ari Csilla , D’Agostino Dominic Paul , Bharwani Sahil , Rehsi Anand , Moss Sara , Schmer-Galunder Sonja , Fiore Stephen 
Published: April 2020

The objective of this study was to measure potential changes in cognitive function in response to living in an extreme underwater habitat. Chronic high stress and intense workload can affect individual and team cognition. As such, associated variables were monitored during the NASA Extreme Environment Mission Operation (NEEMO) 23 mission at the Aquarius reef Base in June 2019.Individual cognition was measured on 6 subjects (2 males, 4 females) with 8 tests using a battery of cognitive assessments (Joggle Research platform), 1 day pre‐mission, at mid‐point of the mission and 1 day post‐mission. In addition, 4 cognitive tests were applied on 5 subjects (2 males, 3 females) at the beginning of Extravehicular Activities (EVA) and at the end of EVAs that lasted for ~3 to 5 hours. To monitor changes in stress level, Short Stress State Questionnaire (SSSQ) and Generalized Anxiety Disorder (GAD7) scores were collected. Effects of workload on performance were measured by NASA Task Load Index (TLX). Changes in team cognition was monitored using an assessment of cognitive process measures.There was a trend of increasing performance in individual cognition in‐habitat and post mission, compared to baseline. The NBACK test, a measure of working memory, significantly improved during the mission, compared to pre‐mission and it further improved post‐mission. The psychomotor vigilance test (PVT) results were non‐significantly lower during the mission. The Motor Praxis Task (MPT) scores decreased at the beginning of EVA and further decreased by the end of EVA. The end of EVA MPT performance was significantly lower, compared to baseline, in habitat, and post mission measurements, suggesting that long duration extravehicular activity had a significant negative effect on sensory motor speed. BART scores were significantly lower at the beginning of EVA, compared to baseline, suggesting that risk taking decreased with the start of the EVA. The PVT score was significantly lower at the end of EVA, compared to baseline, in habitat, and the beginning of EVA, suggesting that the long duration EVAs had negative effect on the ability to quickly respond to the sudden appearance of a stimuli. A trend of increasing GAD7 scores at half time of the mission and post‐mission could be observed. There were differences in NASA TLX and team cognition scores between subjects and at different time points.The present results suggests that sub‐components individual and team cognitive functions may be differently affected during this extreme environment mission. Further research is needed to isolate which cognitive functions might be altered and how these may be correlated with stress level and workload to affect mission success.Support or Funding InformationKetone Technologies LLC provided partial funding for the experiments. SIFT provided partial salary for CA.
Structure‐Based Design and Evaluation of Peptidomimetic Inhibitors Against the Human Norovirus Protease
Authors: Muzzarelli Kendall Marie , Brunzelle Joseph , Kovari Iulia , Yang Zhe , Kovari Ladislau 
Published: April 2020

Norovirus is the leading cause of acute gastroenteritis worldwide and can lead to severe chronic infection in immunocompromised patients. Currently, there are no FDA approved therapeutics or vaccines against Norovirus infection. However, progress has been made in terms of identifying the Norovirus protease as a promising drug target due to its necessity in the production of mature viral proteins. Our study has focused on the development of targeted covalent inhibitors against the human strains of Norovirus protease by employing both computational and enzymatic design and evaluation techniques. Analysis of our recently solved apo and complexed crystal structures of Norovirus GI.1 and GII.4 proteases provided additional insights into the binding pocket and active site composition, which led to improved inhibitor efficacy. Potential compounds were designed and analyzed using Schrodinger (2019‐1) software, allowing for predictive ADME screening, covalent docking of inhibitors to the protein structure, and binding affinity analysis. The lead compounds were synthesized, then enzymatically screened against purified Norovirus protease using a FRET‐based assay, following in silico design and evaluation. To date, our lead compounds exhibit nanomolar activity against the Norovirus GII.4 protease as well as the less infectious GI.1 strain.Support or Funding InformationVera Fay Righthand Fellowship in Virology