bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of High‐Fat Diet on Body Composition and Autonomic Modulation in Rats
Authors: de Souza Campiello Talarico Ana Laura , Simões Barbara Bim , Rozza Caroline Altenburg , Brognara-Dias Fernanda , Dias Daniel Penteado Martins 
Published: April 2020

Currently, more than a third of the world’s population is overweight or obese. In addition, obesity increases the risk of myocardial infarction, heart failure and ventricular remodeling, directly affecting the cardiocirculatory system. The consumption of different types of fats may favor the onset of these cardiovascular diseases. Thus, the present study compared the effects of high‐fat diets with different compositions (predominance of saturated or unsaturated fats) on body composition and cardiovascular parameters of rats. Wistar male rats were assigned into groups and fed with control (standard rat chow), saturated high‐fat diet [standard rat chow enriched with grated raw coconut (1:1)] or unsaturated high‐fat diet [standard rat chow enriched with crushed roasted almonds (1:1)] during eight weeks. The body weight was evaluated twice a week during the whole feeding phase. At the end of this phase, all rats were anesthetized with a ketamine/xylazine mixture and implanted with an electrode for electrocardiogram (ECG) recording and a catheter into the femoral vein for drugs administration. Two days later, unanesthetized animals had the ECG recorded during 30 minutes at baseline, followed by atropine (1 mg/kg, i.v) and propranolol (2 mg/kg, i.v) administration, separated by a 15 minutes interval. On the next day, rats were subjected to the same protocol, but autonomic blockers were given in opposite order. Right after ECG data sampling ceased, animals were fully anesthetized and had the left lobe of the liver, epididymal and retroperitoneal adipose tissues (on both sides) collected and weighed. Despite the difference in diet composition, animals showed comparable weight gain in all groups over time. No difference was observed on the liver weight among groups. However, animals fed with unsaturated enriched diet showed a strong trend towards an increase in the weight of retroperitoneal adipose tissue (control diet: 6.7 ± 1 g; saturated diet: 7.7 ± 1 g; unsaturated diet: 12.9 ± 3 g). Also, both groups receiving high‐fat diets showed a trend towards augmented epididymal adipose tissue weight (saturated diet: 8.2 ± 1 g; unsaturated diet: 10.9 ± 3 g), as compared to control group (control diet: 5.8 ± 1 g). Concerning the autonomic modulation, baseline heart rate and the intrinsic heart rate were comparable among groups studied. Nevertheless, a trend towards reduced vagal tone was seen in unsaturated diet group (control diet: Δ114 ± 24 bpm; saturated diet: Δ121 ± 20 bpm; unsaturated diet: Δ86 ± 11 bpm). In conclusion, although the composition of the diet did not influence the weight gain of the rats over the eight weeks, the current data indicate that the body composition, especially adipose tissue weight, may differ depending on diet basis. In addition, the unsaturated diet seems to decrease vagal tone in rats.Support or Funding InformationBarao de Maua University Center: 347/19; FAPESP: 2017/05163‐6
Integrating Genetic Fine‐mapping in Outbred Rats with a Novel Human Tubule Fibrosis Model Identifies Sept8 as a Gene Involved in Cellular Structure and Kidney Fibrosis
Authors: Woods Leah Solberg , Prokop Jeremy , Keele Gregory , Holl Katie , He Hong , Littrell John , Deal Aaron , Valdar William , Garrett Michael 
Published: April 2020

Chronic kidney disease (CKD), which can ultimately progress to kidney failure, is influenced both genetics and the environment. Genes identified in human genome wide association studies (GWAS) explain only a small proportion of the heritable variance, suggesting the need for additional model systems to identify CKD genes. We performed GWAS for urinary protein and serum biochemistries in male heterogeneous stock (HS) rats. Quantitative trait loci (QTL) were identified using a linear mixed effect model of imputed genotypes which accounts for unequal relatedness between rats. RNAseq and bioinformatics tools were used to identify candidate genes. We then developed a novel human tubule hypoxia induced fibrosis model to test these candidates. We identified two QTL for urinary protein and five for serum biochemistries. Protein modeling identified a missense variant within Sept8 as a proteinuria candidate. We demonstrate increased expression of Sept8 in the in vitro fibrosis model. We further demonstrate that SEPT8 localizes to the cilia in tubule cells under normal conditions and relocalizes outside the cilia after fibrosis, suggesting a role of SEPT8 in cellular organization and structure in response to environmental stress. In human biopsies, we show that SEPT8 expression levels increase with fibrosis levels, providing additional support for this gene in kidney fibrosis. This work suggests that integration of a rat genetic model with an environmentally induced cell culture system of tubule fibrosis has the power to elucidate aspects of the complex gene by environmental interactions that contribute to CKD risk.Support or Funding InformationR01 DK088975 and R01 DK106386 (LSW) and NIH/NHLBI R01HL137673 (MRG)
New Metabolite Agonists and Allosteric Modulators for Understudied “Dark” G Protein‐Coupled Receptors
Authors: Isom Daniel , Kapolka Nicholas , Rowe Jacob , Taghon Geoff , Morgan Will 
Published: April 2020

There are more than 100,000 known human metabolites, but less than 20 known metabolite‐sensing G protein‐coupled receptor (mGPCRs). This is surprising given that more than 800 GPCRs comprise the largest class of cell surface receptors in humans. Here, we confirm our hypothesis that there are more mGPCRs to be discovered by reporting new metabolite agonists and allosteric modulators for several understudied “dark” (GPR4, GPR65, GPR68, and HCAR3) and well‐known (ADRA2A, ADRA2B, MTNR1A, LPAR1, LPAR4, S1PR1, and S1PR2) receptors. We identified these interactions using our new ligand discovery technology, dynamic Cyan induction by Functional Integrated Receptors (dCyFIR), to screen a library of 320 metabolites against an exploratory panel of 300 GPCR‐Gα subunit coupling combinations covering 30 receptors. Our findings support the emerging view that numerous mGPCRs are distributed throughout the body to detect global (endocrine) and local (paracrine and autocrine) metabolite signals, and represent an untapped source of pharmacotherapeutic potential.Support or Funding InformationThis work was supported by R35GM119518, R01NS103906, and R03TR002908 to D.G.I.
WNK1 Regulates Cell Volume via Crowding‐Induced Phase Transitions
Authors: Boyd-Shiwarski Cary R. , Shiwarski Daniel J. , Weaver Claire J. , Beacham Rebecca T. , Griffiths Shawn E. , Subramanya Arohan R. 
Published: April 2020

When challenged by hypertonicity, dehydrated cells recover their volume though regulatory volume increase (RVI). This process is thought to be initiated by an undefined intracellular sensing and transduction mechanism that responds to macromolecular crowding (Parker JC AJP 1993). In many cell types, RVI is mediated by the bumetanide‐sensitive cotransporter NKCC1, which is phosphorylated and activated by the WNK‐SPAK/OSR1 pathway. Reconciling the mechanism of hypertonic stress‐induced activation of NKCC1 has been challenging, as upstream WNK kinases such as WNK1, though required for bumetanide‐sensitive RVI (Roy A AJP 2015), should be inactivated by the strengthened intracellular chloride concentration caused by cell shrinkage (Piala AT Sci Signaling 2014). We describe our efforts to unravel this paradox using a combination of high‐speed live cell and fluorescence lifetime imaging, correlative light electron microscopy, gene editing, and biochemical approaches in cells. During hyperosmotic cell shrinkage and macromolecular crowding caused by reduced cell water, WNK1 dynamically concentrates into membraneless liquid droplets. These changes in WNK1 localization occur at native levels of expression and are visible within physiologic range (25 mOsm above isotonicity), triggering a cascade of condensed WNK1 autoactivation, droplet fusion, cytosolic exchange, and downstream SPAK/OSR1/NKCC1 phosphorylation within a 15 minute bumetanide‐sensitive RVI time course. When cells are rapidly switched from hypertonic to hypotonic media, the WNK1 condensates quickly dissipate, confirming their reversible nature and association with physiologic changes in cell size and intracellular crowding. An optogenetic screening assay that employs the PHR domain of Cry2 to trigger light‐induced crowding and phase separation revealed that the formation of WNK1 condensates requires its large intrinsically disordered C‐terminus, whose strong regional disorder tendency is highly conserved across evolution despite poor sequence homology. This disorder‐encoded phase behavior allows WNK kinases to react to a crowded cytosol, bypass the ionic strength dilemma, and restore cell volume via mass action. Thus, our findings indicate that in addition to their well‐appreciated role as chloride sensors, WNK kinases are molecular crowding sensors that phase separate to coordinate a conserved hyperosmotic stress response. While this evolutionarily conserved mechanism appears to be critical for WNK kinases to sense cell shrinkage and amplify the NKCC1 RVI signal, we propose that it also impacts diverse WNK‐dependent signaling processes important in mammalian epithelial and neuronal physiology.Support or Funding InformationNIH R01DK098145, R01DK119252, K08DK118211, F32HL142229, P30DK079307
Role of Src homology/collagen adaptor protein p66Shc in Pyk2 translocation into mitochondria under Gq protein–coupled receptor stimulation
Authors: Dileepan Gayathri , Polina Iuliia , Cypress Michael , Suzuki Yuta , Adhikari Neeta , Jhun Bong Sook , O-Uchi Jin 
Published: April 2020

IntroductionMitochondrial Ca2+ (mtCa2+) uptake via a main mtCa2+ channel, mtCa2+ uniporter (MCU), mediates cell survival and death via its contribution of mitochondrial ATP synthesis and apoptotic signaling activation, respectively. Our group previously showed that Gq protein–coupled receptor (GqPCR) stimulation promotes activation of a tyrosine kinase named proline‐rich tyrosine kinase 2 (Pyk2), and its translocation from cytosol to the mitochondrial matrix, which leads to the tyrosine phosphorylation of MCU, and mtCa2+ overload, and activation of apoptotic signaling in the cardiomyocytes. However, it is still unclear how Pyk2 access from cytosol to mitochondrial matrix under GqPCR stimulation. The Src homology/collagen (Shc) adaptor protein, p66Shc, is also known to be capable to translocate to the mitochondria where it promotes increased ROS production and apoptosis. Importantly, p66Shc can bind to another tyrosine kinase Src, but its association to Pyk2 has not been well investigated.Hypothesisp66Shc serves as an important chaperon for Pyk2 translocation into the mitochondrial matrix.Aimto test whether p66Shc can associate with Pyk2.MethodsMammalian expression plasmids containing p66Shc or Pyk2 were transformed in to bacteria and purified. Overexpression of p66Shc and/or Pyk2 in HEK293T cells were performed by transient plasmid transfection. Whole cell lysates were collected and used for biochemical assay including Western blotting and co‐immunoprecipitation assay.ResultsWe confirmed that transient transfections of the plasmids containing His‐tagged p66Shc and GFP‐tagged Pyk2 were successfully yield overexpression of these proteins in HEK293Tcells. Co‐immunoprecipitation assay using GFP antibody showed that p66Shc and Pyk2 forms complex in the whole cell lysates from cells co‐transfected with two plasmids.Conclusionp66Shc can bind Pyk2 and may contribute to the mechanism for Pyk2 translocation to mitochondria. Pur next plan is to test whether the removal (overexpression) of p66Shc blocks (Pyk2 transportation and prevent excessive calcium uptake by the MCU under GqPCR stimulation both in heterologous expression system and primary cardiomyocytes.Support or Funding InformationApart of this research was supported by the Lillehei Heart Institute Summer Research Fellowship (to G.D.), American Heart Association (AHA) 18CDA34110091(to B.S.J), NIH/NHLBI R01HL136757 (to J.O.‐U.), AHA 16SDG27260248 (to J.O.‐U.), and American Physiological Society (APS) 2017 Shih‐Chun Wang Young Investigator Award.
The Impact of Dietary Sodium on Sympathetic and Blood Pressure Reactivity Assessed using the Cold Pressor Test
Authors: McMillan Ronald K. , Babcock Matthew C. , Migdal Kamila U. , Robinson Austin T. , Watso Joseph C. , Stocker Sean D. , Wenner Megan M. , Farquhar William B. 
Published: April 2020

High dietary sodium intake increases sympathetic and blood pressure (BP) reactivity in rodents, but it is not known if dietary sodium similarly affects reactivity in non‐hypertensive humans. Importantly, exaggerated sympathetic and BP responses are predictive of future hypertension risk.PURPOSEWe hypothesized that high dietary sodium would increase sympathetic and BP reactivity in male and female adults. The cold pressor test (CPT) was used to assess reactivity.METHODSTwenty‐seven healthy, non‐hypertensive adults (16M/11F; age: 26±7yrs; BMI: 24.7±1 kg/m2; mean±SD) participated in a controlled feeding study that consisted of 10 days of low (LS: 1.0 g/day), recommended (MS: 2.3 g/day), or high (HS: 7.0 g/day) sodium diets, in random order with at least a one‐month washout between diets. Twenty‐four hour urine samples were collected and sodium excretion was calculated to ensure compliance on the respective diets. Muscle sympathetic nerve activity (MSNA) was directly assessed using peroneal microneurography and beat‐to‐beat BP was assessed using photoplethysmography at rest and throughout the CPT. The CPT was completed by having participants immerse their dominant hand in an ice slurry (3° to 5°C) for 2 minutes. Data were analyzed using two‐way repeated measures ANOVAs.RESULTSAs expected, twenty‐four hour sodium excretion increased with increasing amounts of dietary salt (LS: 42±40, MS: 84±36, HS: 253±111 mmol/24 hours, main effect of diet, p<0.0001), with no apparent sex differences (p = 0.98). The CPT robustly increased MSNA, but there was no effect of diet (Δ MSNA burst frequency LS: 13±6, MS: 16±6, HS: 15±5 bursts/min, p = 0.23). Similarly, the CPT acutely increased systolic BP, but there was no effect of diet (Δ systolic BP LS: 18±11, MS: 20±12, HS: 18±11 mmHg, p = 0.44).CONCLUSIONThese data suggest that 10 days of high dietary salt intake does not affect MSNA or BP responses to the CPT in young, healthy, non‐hypertensive adults.Support or Funding InformationSupported by NIH Grant 1R01HL128388
Survival of Mature Dendritic Cells Dependent on Clusterin Expression
Authors: Mendoza Roy Alexander , Malizia Alvaro Lopez , Garcia Guadalupe Ailin , Sabattê Juan Antilio 
Published: April 2020

Clusterin is a multifunctional glycoprotein found in various tissues and body fluids. It is involved in a number of physiological and pathological processes such as apoptosis, protein homeostasis, cancer and Alzheimer’s disease. Although clusterin expression in myeloid cells has been reported, its influence in dendritic cell (DC) function has yet to be analyzed. Therefore, the purpose of our experiment was to determine the role of clusterin in DC survival. First, we analyzed whether activation of human monocyte‐derived DCs resulted in clusterin production. This was done by culturing cells for 48 hours with or without LPS (10 ng/ml). Clusterin was analyzed by western blot and quantified by ELISA. A basal expression level of clusterin was observed in unstimulated DCs, while an increase in production was noted upon activation of these cells by LPS: clusterin concentration in cell supernatant was 0.04 ± 0.1 vs 7.47 ± 2.14 ng/ml (control vs LPS, mean ± ES, n=5, p<0.01 vs unstimulated cells). In order to understand the dynamics of production after activation, we constructed a production curve measuring the level of clusterin post stimulation over a period of four days. A peak in the production of intracellular and secreted clusterin was noted 48 – 72 hours post stimulus. Finally, a knockdown (KD) strategy using a lentiviral gene silencing protocol that delivered a clusterin and scramble (SCR) shRNA was used to determine the role of clusterin in DC function. Two lentiviral combinations were used due to their ability to knockdown >85% of clusterin expression, as evaluated by western blot (in HeLa cells) and rtPCR (DCs). Puromycin was used as a selection drug and a Ficoll gradient was used to separate live and dead cells. We found that silencing clusterin expression (CLU‐KD) resulted in a massive death of DCs upon stimulation with LPS, as evaluated by anexin‐V/propidium straining: 63.7 ± 11.8, 19.7 ± 4.9, and 25.2 ± 6.5% of dead cells, for LPS‐stimulated CLU‐KD DCs, LPS‐stimulated scramble DCs, and unstimulated DCs, respectively (mean ± ES, n=4, p<0.01 CLU‐KD vs scramble or unstimulated DCs). These observations uncover a novel function of clusterin as a key player in the control of the adaptive immune response.Support or Funding InformationRoy Mendoza was supported by the grant LA Basin Minority Health and Health Disparities Research Training Program (MHRT) T37MD001368 from the National Institute on Minority Health and Health Disparities, National Institute of Health.
Liver fibrosis and biliary proliferation are enhanced in the rat model of inflammatory bowel disease
Authors: O’Brien April , Chen Lixian , Cromer Walter , Alpini Gianfranco , Glaser Shannon , Zawieja David 
Published: April 2020

BackgroundPatients who suffer from Inflammatory Bowel Disease (IBD) often experience concurrent conditions such as lymphatic dysfunction and bone loss. Additionally, liver and biliary abnormalities are frequently observed, including primary sclerosing cholangitis (PSC), autoimmune hepatitis, and cholelithiasis. The pathogenesis is unknown but is presumed to be secondary to chronic inflammation, which drives intrahepatic inflammation and fibrogenesis. The lymphatic system, via transport of inflammatory stimuli, is a likely route by which the regional inflammatory changes in the bowel effect distal organs such as the liver. Therefore, the aim of this study was to evaluate changes in biliary damage and senescence, ductular reaction (DR), and liver inflammation and fibrosis in an experimental rat model of inflammatory bowel disease.MethodsStudies were performed in male rats receiving a 2,4,6‐trinitrobenzene sulfonic acid (TNBS) enemas at 30mg/kg of body weight, once a week, for 4 weeks. Lymph, livers, isolated cholangiocytes, and hepatic stellate cells were collected. Ductular reaction was evaluated by immunohistochemistry (IHC) for CK19 in paraffin embedded liver sections and cellular senescence was determined by SA‐β‐gal staining. Gene expression was measured using qPCR for p16, CCL2 and p21 in total liver and laser‐captured microscopy (LCM) isolated cholangiocytes. Liver fibrosis was assessed via Sirius Red staining and qPCR for the following genes; α‐SMA, TGFβ‐1, FN‐1, and collagen1a1 in total liver, HSCs, and pure cholangiocytes. To determine the level of inflammation, IHC for CD68 and MHCII was performed, while gene expression was measured using qPCR for inflammatory/senescence markers (IL‐1β, IL‐6, IL‐33 and TNF‐α) in total liver and LCM isolated cholangiocytes. Immunofluorescence was used to visualize the expression of TLR4 in the intact bile ducts of total liver sections. Lymph was used for proteomic analysis post‐instillation at peak inflammation, to identify potential inflammatory signals that may precipitate the changes that were noted.ResultsRats treated with TNBS had increased DR, collagen deposition, and fibrotic gene expression in both cholangiocytes and HSCs; enhanced liver inflammation with increased CD68 positive macrophages in liver sections; increased biliary senescence and biliary expression of senescence markers; and increased biliary expression of TLR4. There was a strong correlation between the increase in inflammatory pathways in the lymph from IBD animals, with marked increases in pathways associated with bacterial translocation and lipid metabolism.ConclusionWe observed increased biliary ductular reaction that was associated with increased liver inflammation/fibrosis similar to the co‐morbidity of PSC reported in IBD patients.Support or Funding InformationNIH RO1 DK110035
Bioactive properties of protein hydrolysate of cottonseed byproduct: antioxidant, antimicrobial, and angiotensin‐converting enzyme (ACE) inhibitory activity
Authors: Egea Mariana Buranelo , de Oliveira Filho Josemar Gonçalves , Lemes Ailton Cesar , Valencia-Mejia Erika , Fernandes Katia Flavia , de Figueiredo Sousa Heloisa Alves , da Silva Edilsa Rosa , Dyszy Fábio H. 
Published: April 2020

Cottonseed meal is a byproduct generated in large amounts resulting from the process of extracting cottonseed oil. The aim of this study was to investigate the cottonseed byproduct hydrolysate produced with Alcalase® (A), Neutrase® (N), or Flavourzyme® (F), and thus, evaluate its the antioxidant activity and angiotensin‐converting enzyme (ACE) inhibitory activity, as well as ability to inhibit the strains of Staphylococcus aureus, and Escherichia coli. The protein hydrolysates (PH) were obtained using thermal pretreatment (CT – control and HT – thermal pretreatment) and enzymatic hydrolysis of protein extract obtained of cottonseed byproduct. The decreasing order of the degree of hydrolysis of protein hydrolysate: HT‐A>CT‐A>HT‐N>CT‐N>HT‐F>CT‐F. The DPPH scavenging activity of the hydrolysates obtained from the Alcalase®, Neutrase®, and Flavourzyme® treatments at the end of the reaction (100 min) were 95.44%, 78.10%, and 62.94% for the HT hydrolysates, respectively, and 88.60%, 70.69%, and 62.06% for the CT hydrolysates, respectively. The ferric reducing antioxidant power of the hydrolysates obtained from the Alcalase®, Neutrase®, and Flavourzyme® treatments at the end of the reaction were 0.47, 0.44, and 0.34 mg TE/mg sample for the HT hydrolysates, respectively, and 0.38, 0.43, and 0.28 mg TE/mg for the CT hydrolysates, respectively. The results of the growth inhibition of S. aureus obtained from the Alcalase®, Neutrase®, and Flavourzyme® treatments at the end of the reaction were 32.38%, 16.66%, and 22.35 for the HT hydrolysates, respectively, and 28.35%, 16.68%, and 17.85% for the CT hydrolysates, respectively. The heat‐pretreated hydrolysates were able to inhibit the growth of S. aureus. The growth inhibition of E. coli was lower when compared to that of S. aureus. The growth was inhibited from 13 to 18 % and was observed for the HT hydrolysates produced using Alcalase® after 20 min of reaction. The hydrolysates showed ACE inhibition of 74 to 99 %. The hydrolysate produced using Alcalase® with thermal treatment showed higher antioxidant activity and ACE inhibition (99.5%) than other enzymes. In this study, it was possible to obtain PH with different bioactivity properties using adequate processing by successfully choosing proteolytic enzymes and thermally pretreating proteins from the cottonseed byproduct.Support or Funding InformationThis work was supported of the FAPEG (Process 201610267000530), CAPES (001), CNPq (06/2016), IFB (030/2016) and IF Goiano (Process 23218.000788/2018‐41).