bluuebunny/crossref_metadata_2025_split huggingface dataset:
A single nucleotide polymorphism in DDAH1 leads to decreased nitric oxide production in neonatal cord blood‐derived stimulated lymphoblastoid cells
Authors: Milton Avante Dionne , Almazroue Hanadi , Jin Yi , Neinast Reid , Zender Gloria , McBride Kim L. , Nelin Leif D. , Trittmann Jennifer K. 
Published: April 2020

Bronchopulmonary dysplasia (BPD) is chronic lung disease in preterm infants. Pulmonary hypertension (PH) develops in 25–40% of BPD and is a major contributor to morbidity and mortality in BPD patients. Nitric oxide (NO) is a potent vasodilator and apoptotic mediator made by nitric oxide synthase (NOS). NOS is inhibited by asymmetric dimethylarginine (ADMA). Dimethylarginine dimethylaminohydrolase (DDAH) hydrolyzes ADMA. Translational studies from our group identified a single nucleotide polymorphism (SNP) in the DDAH1 gene, rs480414, that was associated with a decreased risk for PH in BPD patients. Therefore, we aim to determine if the DDAH1 SNP rs480414 affects DDAH1 function in cell culture. Neonatal cord blood specimens were treated with Epstein‐Barr virus to select for B‐lymphocytes and transform into lymphoblastoid cell lines (LCLs) that were genotyped (SNP or wild‐type) at the rs480414 locus. We tested the hypothesis that in LCLs the SNP rs480414 results in a gain of function mutation in DDAH1, which would lead to greater NO‐mediated apoptosis as compared to DDAH1 wild‐type (WT). LCLs (WT, n=3 and SNP, n=3) were stimulated with phorbol myristate (PMA), IL‐4, and IL‐13 for 48 hours, protein was analyzed by western blot analysis for DDAH1, cleaved and total caspase‐3 and ‐8, and β‐actin. RT‐PCR was performed and expression of DDAH1 and iNOS mRNA was evaluated. Cell media was assayed for concentrations of nitrite using a chemiluminescence NO analyzer. Spectrophotometry was used to measure the conversion of ADMA to L‐citrulline. A standard L‐citrulline curve was used to calculate DDAH activity after normalization to protein concentration. LCLs with the DDAH1 SNP had similar levels of DDAH1 protein expression (n=3, p=0.58), similar mRNA expression (n=3, p=0.1), and similar DDAH activity (p=0.35) compared to DDAH1 WT. We found that LCLs with the DDAH1 SNP had similar iNOS mRNA expression (p=0.6), lower nitrite levels (p=0.04), lower cleaved caspase‐3 levels (p=0.04), and similar cleaved caspase‐8 levels (p=0.2) than DDAH1 WT LCLs. Contrary to our hypothesis, these finding suggests that the DDAH1 SNP rs480414 may result in a loss of function mutation, resulting in lower NO production and lower apoptosis in LCLs. This is the first evidence that the DDAH1 SNP rs480414 has an effect on cell function, however we speculate that pulmonary vascular endothelial cells will likely have a different physiology.Support or Funding InformationThis work was supported by NIH‐K08 HL129080.
Adhesion‐GPCR Gpr116 (ADGRF5) is a Regulator of Urine Acidification
Authors: Zaidman Nathan , Tomilin Viktor , Damarla Mahendra , Tidmore Josephine , Pochynyuk Oleh , Pluznick Jennifer 
Published: April 2020

G‐protein coupled receptors (GPCR) are a large and diverse family of integral membrane proteins that recognize a tremendous assortment of extracellular molecules including neurotransmitters, hormones, light and odors. They are a common target of pharmaceutical drug development, and uncovering the function of novel GPCRs in the kidney represents a wealth of untapped therapeutic potential. We previously performed an mRNA screen for novel GPCRs in the kidney to identify promising yet overlooked renal GPCRs. This screen revealed that Gpr116, an adhesion‐class GPCR, is highly expressed in the kidney. To understand the role of Gpr116 in renal physiology, we have taken a multidisciplinary approach. Using transfected HEK293 cells expressing cloned Gpr116, we have validated a monoclonal antibody for Gpr116. In murine kidney, this antibody indicates localization of Gpr116 to intercalated cells (ICs) in the collecting duct of mouse kidneys. This staining is absent in kidneys from targeted knockout (KO) of Gpr116 in renal tubules (Gpr116flox/flox, ksp‐Cre). Additionally, kidney‐specific KO animals have significantly (p<0.02) reduced urine pH (5.66±0.29, n=9) compared to wild‐type (WT; 5.96±0.23, n=15) littermates. Challenging the animals with an acid load (280mM NH4Cl added to drinking water) significantly acidifies WT urine (5.71±0.18, n=14, p<0.03) but does not significantly reduce KO urine pH (5.55±0.20, n=9), suggesting the loss of Gpr116 from α‐ICs is sufficient to maximally acidify urine. KO mice do not have significantly more ICs compared to WT. Interestingly, the loss of acid in the urine is accompanied by a small, but significant (p=0.001), increase in blood pH, and a small, but significant (p<0.05), decrease in pCO2 compared to WT. We conclude that loss of Gpr116 from the nephron causes a primary loss of acid in the urine which results in a mild metabolic alkalosis (“renal tubular alkalosis”) due to reabsorption of HCO3− by α‐ICs. This study establishes a physiologic role of the previously understudied Gpr116 in the murine kidney and demonstrates the scientific potential of future investigations into novel GPCRs.Support or Funding InformationResearch was funded by NIH F32DK116499 (N.A.Z.) and NIH R01DK107726 (J.L.P.).
Calcium‐Dependent Localization of S100A6 in Pulmonary Microvascular Endothelial Cells
Authors: Haldar Barnita , Hamilton Caleb Lucas , Solodushko Viktoriya , Honkanen Richard E. , Scammell Jonathan G. , Cioffi Donna Louise 
Published: April 2020

During inflammation, mediators increase calcium influx into endothelial cells through store‐operated calcium entry (SOCE) channels. Increasing calcium influx through the SOCE current ISOC promotes endothelial barrier disruption through the formation of inter‐endothelial cell gaps. Inter‐endothelial cell gap formation leads to increased permeability. Identifying mechanisms of ISOC inhibition may aid in the development of therapeutic strategies against endothelial permeability‐associated vascular inflammation. Our studies indicate FK506‐binding protein 51 (FKBP51), in conjunction with protein phosphatase 5 (PPP5C), inhibit ISOC and calcium entry‐induced inter‐endothelial cell gap formation. Further, we determined that the calcium‐binding protein S100A6 is necessary for the PPP5C‐FKBP51‐mediated inhibition of ISOC in pulmonary microvascular endothelial cells (PMVECs). In PMVECs, S100A6 interacts with the ISOC channel in a calcium‐dependent manner, i.e., following an increase in cytosolic calcium. However, we do not know whether calcium entry specifically through the ISOC channel or through other SOCE channels that provides the calcium source to promote S100A6 interaction with the ISOC channel.MethodsPMVECs were used for co‐precipitation and immunocytochemistry‐based studies. When cells are treated with rolipram (R) plus thapsigargin (Tg), ISOC and other SOCE channels are activated. However, when cells are treated with Tg alone, ISOC is not activated, but other SOCE channels are activated. The TRPC4 subunit of the ISOC channel was immunoprecipitated and probed for co‐precipitation of S100A6.ResultsFollowing R/Tg treatment, robust co‐precipitation of S100A6 with TRPC4 was observed, whereas following Tg alone only minor co‐precipitation was seen. Immunocytochemistry revealed that in untreated cells S100A6 localization is principally cytosolic. Following R/Tg treatment, but not Tg alone, a distinct membrane‐delimited pool of S100A6 was observed, particularly at sites of cell‐cell contact. Membrane localization was confirmed by co‐localization with wheat germ agglutinin (WGA).ConclusionOverall, our data reveal that calcium entry specifically through the ISOC channel is an important determinant of S100A6 translocation to the plasma membrane promoting interaction of S100A6 with the ISOC channel.Support or Funding InformationNIH HL
R56HL107778
Targeted Dietary Supplementation Improves Mental Performance in Children
Authors: Talbott Shawn , Armstrong Andrea , Massa Michelle , Royston Jessica , McHenry Markham 
Published: April 2020

BackgroundSaffron (Crocus sativus) dried flower stigma is the world’s most expensive spice and has been used in traditional medicine for alleviating depression, stress, anxiety, and insomnia. Saffron stigma contains more than 160 bioactive compounds including lepticrosalides (safranal, crocin, crocetin, picrocrocin) and numerous flavonoids and terpenes that have been associated with relaxation, positive mood, and mental & physical balance. At least seven controlled clinical trials have shown the antidepressant activity of saffron.ObjectiveBuilding on the well‐described “mood” benefits of saffron, our objective was to assess the benefits of saffron stigma combined with complementary brain‐supporting spices (Holy Basil, Rosemary, Clove, and Oregano) on measures of mental focus and mental performance in healthy children who had not been diagnosed with either depression or ADHD.MethodsTen healthy children (ages 6–12 years of age) participated in this study. We used the validated NICHQ Vanderbilt Assessment Scales (National Institute for Children’s Health Quality) that are routinely used by healthcare professionals to help diagnose ADHD in children 6–12 years and are part of the American Academy of Pediatrics (AAP) Resource Toolkit for Clinicians Caring for Children with ADHD (3rd edition, 2019). Parents administered the NICHQ survey before and after 30‐days of supplementation with a multi‐nutrient blend intended to improve mental focus, mood, and stress resilience (Kid’s Mood+; Amare Global). The NICHQ assessment scales have 2 components: Symptom Assessment (in 5 areas: inattention, hyperactivity, defiance, conduct, and anxiety/depression) and Performance Assessment (including school performance on reading, writing, and math; as well as social relationship performance with parents, siblings, and peers including on organized teams).ResultsFollowing 30‐days of supplementation, we found dramatic improvements on assessments of both Symptoms (e.g. focus, attention, mood, listening, tension, and irritation) and Performance (e.g. overall school work, math, reading, writing, and social relationships). All participants (10/10) demonstrated benefits in response to supplementation, with average Symptom scores 29% lower (23.4 pre versus 16.6 post) and Performance scores improved 18% (2.24 pre versus 1.83 post).ConclusionsPrevious human trials in children and teenagers have shown equivalence of saffron to fluoxetine (Prozac) for depression and methylphenidate (Ritalin) for ADHD. This is the first study in a population of normal healthy (“non‐diagnosed”) children showing improvements in not just mental focus attributes, but also mental performance (academically and socially) subsequent to targeted supplementation. This study provides compelling evidence for safe and effective natural approaches as potential first‐line therapy for improving focus, mood, and mental performance in children.Support or Funding InformationThis trial was supported by Amare Global
MLP regulates ENaC Activity in Renal Distal Convoluted Tubule Cells
Authors: Song Chang , Moseley Auriel , Yue Qiang , Al-Khalili Otor , Wynne Brandi , Eaton Douglas 
Published: April 2020

The epithelial sodium channel (ENaC) plays an important role in blood pressure regulation through the fine‐tuning of sodium reabsorption in the distal nephron. We previously demonstrated that ENaC is present in distal convoluted tubule (DCT) cells, and the PIP2‐dependent regulation of ENaC is mediated by the myristoylated alanine‐rich protein kinase C substrate (MARCKS)‐like protein‐1 (MLP‐1). MLP‐1 regulation of ENaC is controlled by MLP‐1 phosphorylation of MLP‐1’s PIP2 binding domain. Here we examine the role of MLP‐1 in the function of regulating ENaC in DCT‐15 cells. We also examined whether ENaC interacts with specific domains of MLP‐1. DCT‐15 cells and Cos‐7 cells were used in our study. To further dissect the mechanism of MLP in regulating ENaC in DCT‐15 cells, we constructed various mutants of MLP. WT is the full length wild‐type protein, whereas S3A contains amino acid substitutions in the effector domain to create a molecule that could not be phosphorylated. The S3D mutant was created to mimic constitutive phosphorylation. The GA mutant had an N‐terminal glycine replaced so that it could not be myristoylated. The single‐channel patch‐clamp recordings were used to measure the channel activity of ENaC channels after transfection, and by Mammalian two‐hybrid assay we examined the interaction sites of MLP‐1 and ENaC. S3A construct of MLP has proved to be particularly effective in regulating ENaC function in DCT‐15 cells compared to other variants, given its higher channel activity. MLP‐1 regulates ENaC by changing the open probability of ENaC but not the protein expression or the number of functional channels in DCT‐15 cells. In a two‐hybrid screen, the N‐terminal (but not the C‐terminal) domain of MLP protein associated strongly with the N‐terminus of gamma ENaC and weakly with the N‐terminus of beta ENaC but not with the alpha subunit in cells.Support or Funding InformationSupported by R01 DK‐110409 to DCE and K01 DK‐115660 and ASN Gottschalk AWARD to BMW
ANGPTL4 Induces TMZ Resistance of Glioblastoma by Promoting Cancer Stemness Enrichment via the EGFR/AKT/4E‐BP1 Cascade
Authors: Tsai Yu-Ting , Ko Chiung-Yuan , Chang Wen-Chang , Hsu Tsung-I 
Published: April 2020

Glioblastoma (GBM) is the most aggressive brain tumor, with a strong invasiveness and high tolerance to chemotherapy. With the current standard treatment combining temozolomide (TMZ) and radiotherapy, glioblastoma can be incurable due to drug resistance. The existence of glioma stem‐like cells (GSCs) is considered the major reason for drug resistance. However, the mechanism of GSC enrichment remains unclear. Herein, we found that the expression and secretion of angiopoietin‐like 4 protein (ANGPTL4) were obviously increased in GSCs. The overexpression of ANGPTL4 induced GSC enrichment that was characterized by BMI‐1 and SOX2 expression, resulting in TMZ resistance in GBM. Furthermore, epidermal growth factor (EGFR) phosphorylation induced 4E‐BP1 phosphorylation that was required for ANGPTL4‐induced GSC enrichment. In particular, ANGPTL4 induced 4E‐BP1 phosphorylation by activating PI3K/AKT and ERK cascades for inducing stemness. To elucidate the mechanism contributing to ANGPTL4 upregulation in GSCs, chromatin immunoprecipitation coupled with sequencing (ChIP‐Seq) revealed that specificity protein 4 (Sp4) associates with the promoter region, −979 to −606, and the luciferase reporter assay revealed that Sp4 positively regulates activity of the ANGPTL4 promoter. Moreover, both ANGPTL4 and Sp4 were highly expressed in GBM and resulted in a poor prognosis. Taken together, Sp4‐mediated ANGPTL4 upregulation induces GSC enrichment through the EGFR/AKT/4E‐BP1 cascade.Support or Funding InformationThis research was supported by the Ministry of Science and Technology of Taiwan (MOST 106‐2320‐B‐038 ‐003 ‐MY2, 107‐2320‐B‐038‐001, and 108‐2628‐B‐038‐005‐)
Tracking the ancestral functions of erythropoietin: neuroprotection & mitochondria
Authors: Soliz Jorge , Losantos-Ramos Karen , Suarez Renato , Arias-Reyes Christian , Laouafa Sofien , Furrer Daniela , Joseph Vincent 
Published: April 2020

It has long been thought that erythropoietin (Epo) is exclusively involved in erythropoiesis, allowing erythroid progenitor cells to survive and mature through their antiapoptotic action. We now know that Epo in mammals has also other functions in the brain, playing key roles in the development, maintenance, protection, and repair of the nervous system. However, the recombinant human Epo (rhEpo) has neuroprotective effects in orthoptera insects (such as grasshoppers), and this effect appears to be mediated by the cytokine receptor‐like factor 3 (CRLF3), raising interesting questions about the evolutionary origin of the Epo signaling pathway and its role in invertebrate species. Taking into account that: 1) Epo in mammals modulates the mitochondrial oxidative phosphorylation and the production of reactive oxygen species (ROS) in several tissues, including the brain; and 2) that insects appeared during a geological period (Cambrian explosion) in which the atmospheric O2 was increasing and required the implementation of antioxidant systems at the cellular level; here we tested the hypothesis that activation of the “Epo‐like” system in the brain domestic crickets (Acheta domesticus) exposed to 6 % of hypoxia during 5 days, modulates mitochondrial functions for preventing against oxidative damages. To do so, we used our oxygraph‐2K system (OROBOROS) that measures the mitochondrial bioenergetics in saponin‐permeabilized tissue of 2 mg weight. Our preliminary results showed that rhEpo increased the survival of domestic crickets exposed to hypoxia by 20%. We showed also that, in normoxic animals, rhEpo increased the mitochondrial O2 consumption rate (OCR), but in hypoxic animals, rhEPO limited the increase of mitochondrial OCR. In parallel, rhEpo significantly decreased the production of ROS in hypoxia. These preliminary results suggest that rhEpo significantly improves cricket’s survival under hypoxia, by promoting a robust antioxidant effect through mitochondria. Our data also suggest that a neuroprotective “Epo‐like” endogenous molecule evolved during the “Cambrian explosion” from a urbilaterian (common to vertebrates and invertebrates) ancestor.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada
Single Prolonged Stress Provokes Depressive‐like Behavior, Anxiety and Impaired Social Interaction in Females with Potential for Early Invention with Neuropeptide Y
Authors: Nahvi Roxanna J. , Nwokafor Chiso , Sabban Esther 
Published: April 2020

Sex contributes to susceptibility of stress‐elicited disorders. Women are at nearly double the risk for developing symptoms of post‐traumatic stress disorder (PTSD), depression, anxiety disorders, and social anxiety or phobia. Despite the higher prevalence of these stress‐triggered disorders, animal models, including the single prolonged stress (SPS) PTSD rodent model, and novel therapies for them are largely understudied in females. Previous studies in males indicate that neuropeptide Y (NPY) has therapeutic potential for stress‐elicited disorders; however none examine its effects in females. Here, we evaluated behavioral manifestations in terms of depressive‐like behavior, anxiety, social interaction, and episodic‐like memory and changes in gene expression in the noradrenergic system in the brain after SPS in females. In addition, we examined the potential therapeutic effects of NPY for these behavioral impairments. Females were exposed to SPS and were immediately infused with intranasal NPY or vehicle in three separate experiments. (1) Stressed females treated with 150μg NPY or vehicle were compared to unstressed controls on forced swim test (FST) and for levels of expression of several genes in the locus coeruleus (LC) 12 days after SPS exposure. (2) We examined stressed females treated with 300μg NPY or vehicle and unstressed controls on the elevated plus maze (EPM) and LC gene expression 7 days after SPS stressors. (3) We evaluated stressed females treated with 600μg NPY or vehicle and unstressed controls on the open field (OF), novel object recognition (NOR), social interaction (SI), and EPM at 7, 13, 19, and 21 days after SPS stressors, respectively. SPS led to increased depressive‐like behavior on the FST and anxiety‐like behavior on the OF and EPM, as well as impaired social interaction. Following FST, the rats displayed elevated tyrosine hydroxylase (TH), CRHR1 and Y1R mRNA levels in the LC, consistent with increased activation of the noradrenergic system. The expression level of these mRNAs was unchanged following EPM, except for Y1R. A higher dose of intranasal NPY tended to have larger anxiolytic effects than the lower dose, however unlike in previously tested males neither dose was significantly different from vehicle. Intranasal NPY did significantly prevent impaired social interaction, as compared to vehicle. There was no effect of SPS or NPY on impairment of memory on the NOR. The results indicate that SPS appropriately elicits PTSD associated behavior and molecular impairments in females. Intranasal NPY has potential for therapeutic intervention with important sex‐specific differences.Support or Funding InformationU.S. Department of Defense (DOD) Award No. W81XWH‐16‐1‐0016 and New York Medical College (NYMC)/Touro Bridge Funding Program 49‐506.
Effects of clindamycin hydrochloride on reversing LPS induced growth inhibition and TNF‐α secretion in human periodontal ligament cells
Authors: Wang Jianhong , Duan Hubin , Qiao Xiaohong , Li Youliang , Wu Yunxia 
Published: April 2020

ObjectiveTo investigate the role of clindamycin hydrochloride on reversing Lipopolysaccharide(LPS)‐induced proliferation inhibition and Tumor necrosis factor‐α(TNF‐α) secretion in human periodontal ligament cells.MethodHuman periodontal ligament cells (HPDLCs) was cultured in vitro and identified by immunohistochemical staining. 4–8 generations HPDLCs were selected for the experiment. Above HPDLCs were divided into control group, LPS group and drug group. In the control group, only Dulbecco's Modified Eagle Medium(DMEM) culture solution was added to each hole. In the LPS group,100 μg/ml LPS was added to DMEM culture solution for each hole.In the drug group, different concentrations(10μg/ml,40μg/ml and 160μg/ml) of clindamycin hydrochloride and 100μg/ml LPS were distributed to different test Wells.MTT and enzymelinkedimmunosorbentassay(ELISA) were used to detect the effects of clindamycin hydrochloride on reversing LPS induced growth inhibition and TNF‐α secretion in human periodontal ligament cells.ResultsMTT results showed that the absorbance of MTT in the LPS group decreased significantly compared with that in the control group. Compared with LPS group, the absorbance of MTT in the drug groups with different concentrations of clindamycin hydrochloride showed a trend of increase. ELISA results showed that the level of TNF‐α in the LPS group increased significantly compared with that in the control group. Compared with LPS group, the level of TNF‐α in the drug groups with different concentrations of clindamycin hydrochloride showed a trend of decrease.ConclusionClindamycin hydrochloride can reverse LPS‐induced proliferation inhibition, which is beneficial to the regeneration of periodontal tissue and the production of new periodontal attachment. It also can significantly inhibit the role of LPS in promoting the secretion of TNF‐α by HPDLCs, thereby reducing the inflammatory response of periodontal tissues, which is beneficial to the treatment of periodontal disease.Support or Funding InformationThis study was supported by Lvliang City Science and Technology Fundation Grant, Lvliang people‘s hospital Fundation Grant and China Postdoctoral Science Fundation Grant (2019T120195)