bluuebunny/crossref_metadata_2025_split huggingface dataset:
Generation of Mice Expressing RFP‐tagged Sodium Phosphate Cotransporter NaPi‐IIa
Authors: Ruminska Justyna , Patti Monica , Vollero Alessandra , Bourgeois Soline , Forster Ian , Hummler Edith , Ponce de Leon Veronica , Biber Jürg , Hernando Nati , Wagner Carsten 
Published: June 2021

Homeostasis of inorganic phosphate (Pi) is essential for various physiologic functions. The sodium phosphate cotransporter NaPi‐IIa is located in the brush border membrane (BBM) of the renal proximal tubule and represents the major renal Pi transporter. The plasma concentration of Pi is determined by factors regulating NaPi‐IIa abundance at the BBM. We are developing a novel mouse model expressing NaPi‐IIa tagged with red fluorescent protein (RFP) to analyze the regulation of the cotransporter at the BBM in real time and space and at the molecular level. We have generated RFP‐NaPi‐IIa constructs containing the RFP at three different locations of the cotransporter, the very N‐terminus, within the N‐terminal tail and within the big extracellular loop. Control experiments in opossum kidney cells and in Xenopus laevis oocytes to analyze the cotransporter polarization, PTH sensitivity, Na+‐dependent cotransport activity and kinetic characterization revealed that the RFP within the N‐terminal tail did not impair any of the analyzed parameters. Upon identification of the proper insertion site, we generated three pairs of transcription activator‐like effector nucleases (TALENs) predicted to cut the Slc34a1 (NaPi‐IIa) gene within the desired sequence. The pair with the highest cutting efficiency was chosen for the generation of RFP‐NaPi‐IIa mice. Consequently, we produced RFP donor DNA containing 5' and 3' homologous arms, as well as the RFP coding sequence. TALENs and the RFP donor were injected into one‐cell stage embryos to generate transgenic mice.This work was supported by NCCR Kidney.CH
The Effect of WNK4 on the Na+:Cl‐ Cotransporter is modulated by Intracellular Chloride
Authors: Bazua‐Valenti Silvana , Chavez‐Canales PhD Maria , Rojas‐Vega Lorena , Gonzalez‐Rodriguez Xochiquetzal , Vazquez Norma , Rodriguez‐Gama Alejandro , Argaiz Eduardo , Plata Consuelo , Garcia‐Valdes Jesus , Ellison David , Hadchouel Juliette , Gamba Gerardo 
Published: June 2021

IntroductionThe effect of WNK4 on the renal Na+:Cl‐ cotransporter, NCC is controversial since several studies in vitro and in vivo have shown negative or positive effects. We hypothesize that both effects coexist and are modulated by the intracellular chloride concentration ([Cl‐]i). Piala et al. (2014) showed that L‐WNK1 is a Cl‐‐sensitive kinase and identified two leucine residues forming the Cl‐‐binding site. These leucines are conserved in WNK isoforms.Materials and MethodsWe used the functional expression system of Xenopus oocytes and HEK293 cells. NCC activity was measured by 22Na+ uptake and/or amino acid threonine phosphorylation in the absence or presence of wild type or mutant WNK4, in control conditions, or after [Cl‐]i depleting maneuvers. WNK4 autophosphorylation was also assessed in these conditions.ResultsAt baseline [Cl‐]i ∼ 45 mM, WNK4 autophosphorylation was undetectable and NCC activity was either inhibited or unaffected. [Cl‐]i reduction (25‐30 mM) promoted autophosphorylation transforming WNK4 to an activator of NCC in a kinase‐dependent manner. The L322F substitution transformed WNK4 into a constitutively autophosphorylated kinase that activated NCC without chloride depletion.ConclusionWNK4 is Cl‐ sensitive kinase exerting differential effects on NCC depending on the [Cl‐]i. It is possible that small changes in [Cl‐]i in distal convoluted tubule cells may have a crucial role determining WNK4 effect on NCC, suggesting that diverse physiological processes could converge in the Cl‐modulation of WNK's.
Effects of NOD2 in intestinal epithelial cells: Cross‐talk between NHE and Na+‐K+‐ATPase activity
Authors: Cabral Jose , Grácio Daniela , Soares‐da‐Silva Patricio , Magro Fernando 
Published: June 2021

NOD‐like receptors are a family of pattern recognition receptors that plays a role in the control of inﬂammation and have been associated with inflammatory bowel disease (IBD). Na+/H+ exchangers (NHE) are responsible for the net uptake of sodium, electrolyte homeostasis and decreases in pHi during inflammatory process.AimEvaluate the effect of NOD2 upon NHE and Na+‐K+‐ATPase activity.MethodsNHE activity, Na+‐K+‐ATPase activity and levels of intracellular calcium were measured using fluorimetric assays.ResultsShort‐term NOD2 activation stimulated NHE1 activity in a concentration dependent manner (100±3 to 129±4% in T84 and 100±4 to 133±2% in NCM460 cells). To understand the transduction signalling involved experiments were performed in the presence of inhibitors of Rip‐like interacting caspase‐like apoptosis‐regulatory protein kinase (RIP2), NF‐kβ, tyrosine‐protein kinase (SRC), transforming growth factor‐beta‐activated kinase1 (TAK 1), TNF receptor associated fator 6, adenylyl cyclase (AC), protein kinase A (PKA), phospholipase C (PLC), protein kinase C, Na+‐K+‐ATPase and potassium channels blockers. The NOD2‐mediated increase in NHE1 activity was prevented by inhibitors of RIP2, SRC, TAK1, AC, PKA, PLC, Na+‐K+‐ATPase and Ca2+‐dependent‐ATP‐sensitive K+ channels in both cell lines; this pathway was NF‐kβ independent. Increases in intracellular Ca2+ were observed after NOD2 activation (100±1 to 360±1% in T84 and 100±1 to 254±12% in NCM460 cells).ConclusionsActivation of NOD2 promotes an increase in Na+‐K+‐ATPase and NHE1 activities as a result of opening of Ca2+‐dependent ATP‐sensitive K+ channels. A better understanding of this pathway is considered for the development of novel therapies for IBD.
BMPR2 mutation in cardiomyocytes impairs insulin‐dependent trafficking of CD36
Authors: Talati Megha , Funke Mitchell , Penner Niki , West James , Hemnes Anna 
Published: June 2021

In PAH, insulin resistance is associated with impaired RV function. In mouse model of PAH with mutant BMPR2, we have shown impaired fatty acid oxidation and lipid accumulation in RV cardiomyocytes. We hypothesize that in cardiomyocytes with mutant BMPR2 insulin dependent translocation of CD36, fatty acid transporter molecule, is impaired and may contribute to lipid accumulation.H9C2 cardiomyocytes were stably transfected with empty vector (control) or plasmid containing mutant BMPR2 (mutant). CD36 protein was analyzed by western and confocal microscopy at baseline (BL) and insulin stimulation (IS) at 15 minutes (m), 30m, 1 hour (h), 24h. Key intracellular insulin signaling pathway intermediates analyzed by western.At BL, CD36 was 2fold up in mutant compared to control and increased 2fold in controls at 1h of IS. In mutant, IS lead to 50% increase in CD36 from 15m to 24h. Immunolocalization studies recapitulated these findings. IS increased CD36 in the cytoplasm and plasma membrane (PM) in mutant compared to control. At BL, pAkt Ser473, a key intermediate, was 2fold up in mutant compared to control. Within 15m of IS there was 4fold increase in pAkt Ser473 in control, and droping to BL by 24h. In mutant, there was similar increase in pAkt Ser473 for 1h, before dropped to BL at 24h. In control and mutant, levels of pAkt Ser473 substrate were similar at BL. In control, IS resulted in 2 and 5fold increase in pAS160 and AS160 from 15 and 30m to 24h respectively. In mutant pAS160 increased 2fold at 15m and AS160 protein increased only slightly compared to BL.Using cardiomyocytes with mutant BMPR2, we show impaired insulin‐mediated Akt‐AS160‐CD36 trafficking resulting in enhanced CD36 translocation to PM. This altered axis may contribute to increased lipid import and cardiac lipotoxicity.1 P01 HL 108800‐01A1
Sec10 and the Exocyst are Necessary for Renal Epithelial Monolayer Homeostasis and Ciliogenesis In Vitro and In Vivo
Authors: Polgar Noemi , Lee Amanda , Lui Vanessa , Fogelgren Ben 
Published: June 2021

The eight‐protein exocyst complex regulates targeted delivery of exocytic vesicles. A previous report demonstrated that shRNA‐knockdown of Sec10 (Sec10‐KD), a central subunit of the exocyst, in Madin‐Darby canine kidney (MDCK) cells disrupted primary cilia assembly and 3D cyst formation. To further study the mechanisms by which Sec10 and the exocyst regulate epithelial polarity, morphogenesis, and homeostasis, we used three dimensional collagen cultures of MDCK cells. Sec10‐KD cysts initially showed undisturbed lumen formation, even though subsequently displayed significantly fewer and shorter primary cilia than controls. Later in cystogenesis, while Sec10‐KD cysts showed numerous apoptotic cells extruded basally into the collagen matrix, control cells maintained normal homeostasis. Sec10‐KD MDCK cells were also more sensitive to apoptotic triggers than controls. Apico‐basal polarity appeared normal in Sec10‐KD cysts, while a planar cell polarity defect was suggested by the differences in mitotic spindle angles between knockdowns and controls. Furthermore, analysis of renal tubules in our recently generated kidney‐specific Sec10 knockout mouse model revealed significant defects in primary cilia assembly and in the targeted renal tubules, abnormal epithelial cell extrusion was also observed, supporting our in vitro findings. We hypothesize that in Sec10‐KD cells, the disrupted exocyst activity leads to an increased apoptotic sensitivity through disrupted primary cilia signaling, and in combination with an increased basal cell extrusion rate, it affects epithelial barrier integrity and homeostasis.Supported by: the NIH, March of Dimes, UH RCMI‐BRIDGES and HRFDCC at University of Alabama.
Hypoxia and Hypercapnia Impairs Cardiac Vagal Control: the Role of Orexin
Authors: Dergacheva Olga , Mendelowitz David 
Published: June 2021

Patients with obstructive sleep apnea (OSA) have an increased risk of cardiovascular diseases and altered plasma levels of orexin. REM sleep further increases the incidence of adverse cardiovascular events. The mechanisms underlying REM sleep‐related reduction in parasympathetic activity include activation of inhibitory input to cardiac vagal neurons (CVNs) in the brainstem originating from the lateral paragigantocellular nucleus (LPGi), a nucleus which plays a role in REM sleep control. This study tests the hypotheses that (1) chronic intermittent hypoxia and hypercapnia (CIHH) inhibits CVNs due to exaggeration of GABAergic pathway from the LPGi to CVNs; (2) orexinergic signaling in CVNs is diminished by CIHH; and (3) identified orexin neurons (orexin‐EGFP‐2A‐Cre rat) are inhibited by acute hypoxia/hypercapnia. GABAergic neurotransmission to CVNs evoked by electrical stimulation of the LPGi was examined using whole‐cell patch‐clamp recordings in rats exposed to CIHH and control rats. GABAergic synaptic events were enhanced following 4 weeks of CIHH. Orexin‐A elicited inhibition of LPGi‐evoked GABAergic current in control animals but evoked no significant changes in CIHH rats. Acute hypoxia/hypercapnia inhibited the firing activity of orexin neurons and diminished glutamatergic neurotransmission to orexin neurons. In summary, repetitive episodes of hypoxia/hypercapnia would diminish activity of orexin neurons, including the orexinergic neurotransmission to CVNs. Diminished orexinergic signaling, along with exaggerated inhibitory neurotransmission from the LPGi to CVNs would reduce cardio‐protective parasympathetic activity and increase risk of cardiovascular diseases.Support: Christian Gillin Research Grant 004GN12 to O.D. and NIH grants HL49965, HL59895 and HL72006 to D.M.
Cerebral Vascular Changes in Space Mice Calvaria
Authors: Siamwala Jamila , Macias Brandon , Healey Robert , Pichika Rajeshwari , Hargens Alan 
Published: June 2021

Spaceflight induces headward fluid shifts which may remodel calvarial bone. Previous microCT studies from our lab showed that mice exposed to 15 days of microgravity had increased bone volume density and cortical thickness of calvaria compared to ground control mice. Bone formation is initiated by angiogenesis during normal bone development. VEGF, a key angiogenic molecule, interacts with BMP2 in bone formation and bone healing. Our hypothesis was that bone formation in space mice calvaria is accompanied by vascular changes.Coronal sections from ground control (GC) and spaceflight (SF) mice (n=2) calvaria were stained with eosin and haematoxylin. Unstained sections were processed using a series of dehydration steps with xylene followed by rehydration with ethanol. The slides were then stained with lectin which binds to blood vessels. The vessels were counted manually from 6 sections (10 fields/slide, n=3). BMP‐2, VEGFA and iNOS gene expressions were quantified using real time PCR.Our results document no difference in the number of vessels between GC and SF mice. However the diameter of the vessels (4 vessels/slide) measured using image pro was significantly lower in SF compared to GC mice (34.1 bfl vs 60.3 bfl, p<0.0001, t test). BMP‐2 expression increased 11 fold in SF (n=6, p<0.01, t test). However there was no significant change in VEGFA. Further there was iNOS gene expression in SF (0.3) compared to GC mice (1). These results corroborate that vascular changes observed in the space flight mice maybe partly responsible for increased bone observed in the SF mice. Future studies are required to study the mechanisms of bone‐vascular interaction and bone growth. Supported by Wood Whelan fellowship (JHS), NASA grants NNX09AP11G and NNX13AJ12G (ARH), NSBRI through NASA NCC 9‐58 (BRM).
CALCIUM SENSING RECEPTOR DELETION IN THE MOUSE ESOPHAGUS ALTERS THE EXPRESSION OF CELL‐CELL JUNCTIONAL PROTEINS
Authors: Abdulnour‐Nakhoul Solange , Tu Chia‐Ling , Que Jianwen , Islam M Toriqul , Brown Karen , Nakhoul Nazih 
Published: June 2021

Calcium sensing receptor (CaSR) is the molecular sensor by which cells respond to small changes in extracellular Ca+2 concentration. It regulates Ca+2 homeostasis by modulating PTH secretion and Ca+2 reabsorption in the kidney. CaSR also plays a role in glandular and fluid secretion in the GI tract and regulates differentiation and proliferation of keratinocytes. CaSR is present in the esophageal epithelium, but its role in this tissue has not been defined. We deleted CaSR in the mouse esophagus by generating KRT5‐CreER;CaSRloxP/loxP compound mutants, where loxP sites flank exon 7 of CaSR gene. For lineage tracing of epithelial cells we bred‐in the reporter allele Rosa26R. Recombination was initiated with multiple tamoxifen injections. We demonstrated exon 7 deletion and Cre expression by PCR analysis of genomic DNA and LacZ staining. qRT‐PCR & Western blot (WB) analyses showed a significant reduction in CaSR mRNA & protein expression as compared to control CaSRloxP/loxP mice. Microscopic examination of esophageal tissues (H&E) did not show gross abnormalities. However, WB data revealed a significant reduction (~25%) in levels of adherens junction proteins E‐cadherin and β catenin and tight junction protein claudin‐1. Levels of Rac/Cdc42, small GTPase proteins involved in actin remodeling, were also reduced. Our data indicate that CaSR plays a role in regulating cell‐cell junctional complexes and is therefore important for the maintenance of the barrier properties in the esophagus.Support: VA Merit & Tulane Enhancement Award
Cinobufotalin Inhibits Ovarian Cancer Cells Proliferation, Migration and Invasion
Authors: Afroze Syeda , Co Mary Antonette , McDowell Anthony , McCormick Timothy , Beeram Madhava , Zawieja David , KUEHL Thomas , Uddin Mohammad 
Published: June 2021

ObjectiveCinobufotalin (CINO), a cardiotonic steroid, is extracted from the skin secretions of the traditional Chinese medicine giant toads (Chan Su). CINO has been used as a cardiotonic, diuretic and a hemostatic agent. Recently, it has been shown that CINO inhibits the lung cancer cells function. In the present study, We evaluated the effect of CINO on ovarian cancer, SK‐OV‐3 cell function in vitro.Study DesignCells were treated with 0.1, 1, 5, and 10 μM CINO. Cell proliferation, migration and invasion were measured using kits. The effect of CINO on p38 phosphorylation was evaluated by a cellular activation of signaling ELISA kit. Cell cycle arrest and apoptotic signaling were determined by fluorescence‐activated cell sorting (FACS) analysis.ResultsCINO at 蠅 0.5 μM inhibited CTB cell proliferation, migration, and invasion (p < 0.05) but had no effect on cell viability. There was a higher (p < 0.05) percentage of G0/G1 phase cells in groups treated with CINO at 蠅 0.5 μM. CINO caused an increase in stress signaling p38 MAPK and a positive annexin‐V staining in SK‐OV‐3 cells, indicating the activation of apoptotic signaling.ConclusionThis data demonstrates that CINO inhibits SK‐OV‐3 cell function via cell cycle arrest and apoptotic signaling. The results of this study suggest that CINO might be further investigated as a novel anti‐ovarian cancer agent.
Efficient Isolation, Expression and Improvement Technique System for Feed Enzymes in China
Authors: Yang Peilong , Yao Bin 
Published: June 2021

Supplementation of hydrolytic enzymes in feed can decrease the effect of anti‐nutritional factors and increase feed conversion rate by 5‐10%. Consequently, this practice can improve the efficiency of livestock production and reduce environmental contamination. The authors have developed a highly efficient platform to enrich the genetic resources of several enzymes, characterize and assess their application potentials, and achieve high‐yield production. These works make the production of novel enzymes cost‐effective and commercially valuable.Firstly, a new technology system was set up, which can directly clone full‐length genes and screen objective genes from environmental metagenomic or transcriptomic nuclear acid. Using these methods, 184 genes coding for enzymes have been identified in short time. The enzymes showed different properties including high catalytic efficiency, thermophilic, acidophilic, or protease‐resistance. Secondly, great contributions were made in the structure and function relationship research of enzyme proteins. An efficient screening vector system was constructed based on extracellular fusion mutant proteins, optimized the protein engineering system for enzyme improvement, and obtained several improved enzymes with excellent properties and application potentials. Third, another great finding was identification of several key factors in Pichia expression system and verification of their function. Based on these knowledge, a high‐yield expression system of 5‐50 mg/mL was developed for large‐scale, cost‐effective production of enzymes, including phytase, xylanase, glucanase, mannannase, and α‐galactosidase.
Microbial Community Responses to Toxic Hydrocarbon Exposure
Authors: Morais Daniel , Pylro Victor , Tótola Marcos 
Published: June 2021

The microbial community within each living organism is essential for its healthy state. Even highly complex matrices like soil, depend on microorganisms to sustain and even improve plant development. Although it is a very robust and well‐equilibrated system, soil microbial populations are prone to changes of abundance caused by environmental changes. These community shifts may result in loss of groups related to biogeochemical cycles or soil structure. This work aimed to compare changes in soil microbial community driven by the amendment with crude oil. We used microcosms of two soils from Rothamsted Research UK agricultural station, being one a bare fallow soil, one a grassland soil and a pristine beach soil (tide line under Cyperus atlanticus growth) from Trindade Island ‐ Brazil. Soils were contaminated with crude oil at a concentration of 5% (w/w) and incubated at 26 °C for 20 days. We monitored CO2 emissions and extracted total DNA and total RNA. The DNA obtained was used for microbial profiling using the protocol suggested by Earth Microbiome Project (http://www.earthmicrobiome.org). The bioinformatics pipeline was executed as recommended by the Brazilian Microbiome Project (http://www.brmicrobiome.org). The preliminary results showed that the three environments are highly divergent about its microbial community even when they presented very similar CO2 emissions. The pristine soil presented more archeal individuals and a bigger community shift between the treated and non‐treated state, a sign of fragility. As future research we intent to evaluate the expression of genes related to soil services and related to degradation of crude oil hydrocarbon.We are thankful to Rothamsted Research for the support and to our funding agencies CAPES and CNPq.
Suppression of ACSL6 in Skeletal Muscle Induces Alteration of Glucose Metabolism
Authors: Bu So Young 
Published: June 2021

Understanding the role of various sources of fatty acids and genes involved in fatty acid metabolism provide core information how nutrition therapy can be applied to the people who has different metabolic trait or genetic variance in susceptibility of metabolic diseases. Numerous isoforms of long chain acyl‐CoA synthetase (ACSL) mediate intracellular fatty acids channeling and regulate energy metabolism. However the role of these genes in skeletal muscle need to be identified in order to understand how they contribute to whole body energy metabolism. The purpose of this study was to identify a role of ACSL6 in skeletal muscle energy metabolism. C2C12 mouse myoblasts were cultured and expression of genes that involving glucose and fatty acids metabolism were analyzed. Also siRNA mediated knockdown of ACSL6 was performed to characterize the role of ACSL6 in skeletal muscle. In mouse skeletal muscle cells mRNA and protein expression of ACSL6 were increased over the time course of cellular differentiation. ACSL6 was up‐regulated by high‐glucose but its expression was not influenced by insulin treatment. Knockdown of ACSL6 decreased glucose without affecting insulin signaling or mitochondrial beta oxidation suggesting that ACSL6 is involved in glucose uptake or utilization independent of early stage of insulin signaling. On‐going study to investigate the effect of tissue specific knockdown of ACSL6 on glucose uptake and insulin signaling pathway in animal model will provide further insight into the role of ACSL6 in skeletal muscle.This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A1019253
Anti‐Inflammatory Dietary Intervention Selectively Improves Insulin Sensitivity in Metabolically Unhealthy Overweight Adolescents
Authors: McMorrow AM , Connaughton RM , McGillicuddy FC , Healy ML , Roche EF , Lithander FE , Roche HM 
Published: June 2021

Anti‐inflammatory nutritional approaches may attenuate obesity‐induced insulin resistance; however results from clinical studies are inconsistent. Baseline metabotype may partially discriminate responders from non‐responders.This 8‐wk randomized, placebo‐controlled, cross‐over trial assessed the determinants of inter‐subject variability in response to an anti‐inflammatory supplement containing LC n‐3 PUFA, vitamins C, E and polyphenols in overweight adolescents (n=58; age 15.9±1.6y; BMI 32.1±6.5kg/m2). Subjects who demonstrated >12.5% improvement in HOMA‐IR were considered responders.Anti‐inflammatory supplementation selectively reduced HOMA‐IR in 38% of subjects (responders; supplement ‐33.01±17.84% v placebo 14.57±54.91%, p<0.001). In comparison with non‐responders, responding subjects demonstrated an adverse pre‐treatment metabotype characterized by increased HOMA‐IR, total and LDL cholesterol and soluble CD163 (p<0.05), despite similar BMI. Additionally, changes in adiponectin concentration significantly predicted HOMA‐IR response to intervention (p=0.04). PBMC mRNA expression of adiponectin receptor 2 increased in responders only (p=0.03).Despite similar BMI to non‐responders, the insulin resistant and dyslipidemic phenotype of responders enhanced the impact of the anti‐inflammatory intervention. This illustrates potential efficacy optimization within the context of personalized nutrition.FundingNational Children's Research Centre, Ireland and SFI 11/PI/1117