bluuebunny/crossref_metadata_2025_split huggingface dataset:
Optimal Chemotherapeutic Combination of 9 Putative Natural Compounds from Traditional Chinese Medicine
Authors: Berlin Ian Geddes , Kenealey Jason 
Published: April 2020

Prostate cancer accounts for 9.9% of all new cancer cases in the United States annually. Despite its high average 5‐year survival rate of 98%, if the cancer metastases to a distant site the 5‐year survival rate drops to just 30.5%. Natural compounds are often studied for their potential chemotherapeutic effects or their sensitizing effects to decrease cancer progression. Traditional Chinese medicine is a common source for finding bioactive small molecules which may have chemotherapeutic effects. This study focused on 9 putative natural compounds and their effectiveness of treating PC‐3 human prostate cancer cells. PC‐3 cancer cells were allowed to adhere for 24h incubated at 37°C with 5% CO2 then treated for 48h. Cell viability was analyzed after an additional 6h incubation with AlamarBlue and tested for relative cell viability. IC50s for each compound were determined and then used in Mixture Design Response Surface Methodology (MDRSM) to determine the optimal mixture of compounds. Chou Talalay statistical analysis was used to determine if combination effects were synergistic, antagonistic or additive. The compounds used include; Triptolide, .01819uM (Ttd), Shikonin, .6002uM (Shk), Curcumin 20.83uM (Cur), Emodin, 57.38uM (Em), Wogonin, 97.87uM (Wo) Berberine, 101.4uM (BB), Silibinin, 106.2uM (or Silybin) (Sy), Epigallocatechin gallate, 272.6uM (EGCG), and β‐Elemene, 304.3uM (β‐E). Emodin, Silibinin and EGCG all appeared to act primarily via cell cycle inhibition and their effectiveness was found to increase in combination with other small molecules. The ideal combination provided a multi‐faced approach reduce cell viability which suggests it may help treat prostate cancer cells in vivo either in tandem with classical chemo‐therapeutics or alone.Support or Funding InformationSimmons Center for Cancer Research
P2Y2 Receptor Modulates Leukocyte‐Endothelium Interaction
Authors: Wang Jianjie , Thomas Spencer , Ulbricht Randi , Marino Hailee , Sheravina Sharon , Huxley Virginia , Erb Laurie , Weisman Gary 
Published: April 2020

Nucleotide P2Y2 receptor (P2Y2R) plays important roles in inflammation. The leukocyte‐endothelium interaction is essential for leukocyte recruitment at the site of tissue damage or injury. Little is known about the role of P2Y2R in the regulation of in vivo leukocyte‐endothelium interaction. This study tested the hypothesis that P2Y2R mediates leukocyte‐endothelium interaction using the cremaster muscle. Leukocytes were labeled with Rhodamine 6G injected via tail vein. Leukocyte rolling and stable adhesion were analyzed using a semi‐automatic leukocyte tracking method. P2Y2R activation by UTP increased leukocyte rolling (EC50 = 4.7 ± 1.4 x 10−7 M) and adhesion (EC50= 4.3 ± 1.3 x 10−6 M) in a concentration‐dependent manner in C57BL/6 wild‐type (WT) mice (n=3). UTP (10−5 M) induced sustained increases in both leukocyte rolling and adhesion in WT mice (n=7–19, P<0.05) over 60 min. P2Y2R knockout (P2Y2R−/−) mice exhibited a 2‐fold increase in baseline leukocyte rolling (rolling fraction=37.8%, n=8, p < 0.001) compared to WT mice (rolling fraction=16.2%, n=12) and stayed at the high basal level without changes in rolling following stimulation of UTP (10−5 M) over 60 min. Baseline leukocyte adhesion in P2Y2R−/− mice was 1.5‐fold increase (15 ± 4 adherent cells.100 μm−1, n=12, p < 0.001) compared with WT mice (9 ± 4 adherent cells·100 μm−1; n=19). There was no change in leukocyte adhesion from the baseline in response to UPT (10−5 M) stimulation in P2Y2R−/− mice (n=5–12). We conclude that P2Y2R prevents in vivo leukocyte rolling and adhesion under resting condition while activation P2Y2R by UTP increases both leukocyte rolling and adhesion.Support or Funding InformationMissouri State University
Potential of Aerobic Physical Training to Minimize the Effects of Leptin Deficiency in Female Mice
Authors: Azevedo-Martins Anna Karenina , Boschetti Daniela , Muller Cynthia R. , Américo Anna Laura , Vecchiatto Bruno , Martucci Luiz Felipe , Pereira Renata , Oliveira Cláudia , Evangelista Fabiana 
Published: April 2020

Aerobic physical training (APT) is an important strategy for prevention and treatment of comorbidities such as type 2 diabetes and obesity. Leptin and adiponectin play an important role in energy metabolism, whose imbalance is strongly related to obesity and insulin resistance (IR). Although women have higher levels of leptin and adiponectin compared to men, in conditions such as obesity and/or type 2 diabetes these levels decrease, abolishing the protective effects of these adipokines in females. This study investigated the effects of APT on the metabolic parameters in leptin‐deficient female mice. For this, 8‐week‐old wild C57BL/J6 (C57) or leptin‐deficient ob/ob (Lepob) female mice were separated into the groups sedentary (S) (C57S, n = 6 and LepobS, n = 5) and trained (T) (LepobT, n = 8). The APT was performed during 1 h/day at 60% of maximal speed achieved in the maximal physical exertion test, 5 times/week for eight weeks. The procedures were approved by the Ethics Committee of the School of Arts, Sciences and Humanities of University of Sao Paulo (#001/2017). LepobS and LepobT groups increased body weight, the weight of retroperitoneal (WAT‐RP) and subcutaneous (WAT‐SC) fat pads compared to C57S. Adipocyte area and diameter of WAT‐RP and WAT‐SC were higher in both LepobS and LepobT compared to C57S, however the APT decreased the WAT‐SC adipocyte area and diameter in LepobT group compared to LepobS. LepobS and LepobT groups showed lower weight of pancreas, gastrocnemius and plantar muscles compared to C57S, however APT prevented the mass reduction of gastrocnemius and plantar muscles in LepobT. Food intake was higher in LepobS compared to C57S, which was counteracted by APT in the LepobT group. The LepobS had fasting hyperglycemia (131 ± 1.02 mg/dL) and glucose intolerance (AUC: 31522 ± 1148 mg/dL/120min) compared to C57S (92 ± 2.51 mg/dL and AUC: 20339 ± 1857 mg/dL/120min). Despite of LepobT group had shown glucose intolerance (AUC: 30085 ± 547 mg/dL/120min), the APT decreased the fasting glycemia (114 ± 6.11 mg/dL) compared to LepobS group. Adiponectin level did not change among groups, as well as the relative area of islets in the pancreatic tissue. During the maximal physical exertion test, LepobT showed higher maximal speed, time to exhaustion and speed on the VO2máx compared to LepobS group. In conclusion, APT prevented the loss of gastrocnemius and plantar muscles mass, reduced WAT‐SC adipocyte hypertrophy and food intake, improved fasting glycemia and aerobic capacity in leptin deficient female mice.Support or Funding InformationFAPESP (#2018/22361‐9), CAPES, CNPq
Doxorubicin Cardiotoxicity in Young Tumor‐Bearing Mice
Authors: Abdelgawad Ibrahim Y. , Grant Marianne K.O. , Lewis Christine , Popescu Flavia , Largaespada David , Zordoky Beshay N. 
Published: April 2020

BackgroundChildhood cancer survivors have about 15‐times higher risk of heart failure than their siblings mainly due to the cardiotoxicity of chemotherapy. Nearly 50% of pediatric cancer patients receive doxorubicin (DOX) which is known to be cardiotoxic. DOX‐induced cardiotoxicity had been extensively studied in tumor‐free mouse models. The objective of the current study is to characterize a juvenile tumor‐bearing immunocompetent mouse model of DOX‐induced cardiotoxicity.MethodsJuvenile five‐week old male C57Bl/6 mice were injected subcutaneously with EL4 lymphoma cells (5X104 cells/mouse) in the flank region suspended in sterile PBS, while tumorfree mice were only injected with sterile PBS. One week after tumor implantation, both tumorfree and tumor‐bearing mice were injected intraperitoneally with either a clinically relevant dose of DOX (4 mg/kg/week) or saline for 3 weeks. One week after the last DOX injection, mice were euthanized and the hearts and tumors were harvested and weighed. Gene expression of molecular markers of cardiotoxicity and inflammation was quantified using real‐time PCR.ResultsDOX 4 mg/kg/week for 3 weeks significantly reduced tumor weights in EL4 tumor‐bearing mice. DOX administration caused significant cardiac atrophy in tumor‐free and tumor‐bearing mice. Moreover, tumor itself caused a significant reduction in heart weight compared to tumor‐free mice. Additionally, DOX administration induced the expression of markers of cardiotoxicity, ANP and BNP, in both tumor‐free and tumor‐bearing mice. Saline‐treated tumor‐bearing mice demonstrated higher expression of TNF‐alpha, an inflammatory marker, compared to tumor‐free mice.ConclusionWe have established a juvenile tumor‐bearing immunocompetent mouse model to investigate DOX‐induced cardiotoxicity. Our tumor‐bearing mouse model will optimize our understanding of the interplay between tumor and DOX‐induced cardiotoxicity. Additionally, this clinically relevant model will be used for investigation of cardioprotective strategies that mitigate DOX cardiotoxicity without compromising its chemotherapeutic efficacy before translating to the clinical settings.Support or Funding InformationResearch was supported by the National Institutes of Health’s National Center for Advancing Translational Sciences, grant UL1TR002494, and by a grant from the St. Baldrick’s Foundation [B.N.Z.]. B.N.Z. is a Masonic Cancer Center Women’s Health Scholar, sponsored by the Masonic Cancer Center, and administered by the University of Minnesota Women’s Health Research Program.
Optimization of Cas9 nuclear localization in a tilapia (Oreochromis mossambicus) cell line
Authors: Villapando Angelika Marie , Hamar Jens Carlton , Kültz Dietmar 
Published: April 2020

CRISPR gene targeting studies in cell lines require reliable expression of Cas9 along with appropriate target‐specific guide RNAs. Effective CRISPR/Cas9 gene editing requires Cas9 localization in the nucleus. Previous attempts to implement CRISPR/Cas9 gene editing in a tilapia (Oreochromis mossambicus) cell line (OmB cells) were unsuccessful. A possible contribution to these results could be inefficient nuclear localization of Cas9. Conventionally, the mammalian SV40 nuclear localization signal (SV40 NLS) is used to localize proteins to the nucleus. Our previous studies have shown that the SV40 promoter is poorly recognized in OmB cells. Therefore, we hypothesized that the SV40 NLS will also be poorly recognized in OmB cells. To test this hypothesis we fused the SV40 NLS to the C‐terminus of GFP (green fluorescent protein), transfected it into the tilapia OmB cell line, and determined the efficiency of nuclear GFP localization using fluorescence imaging (Leica Dmi8). Our imaging results show that the SV40 NLS moderately induced nuclear GFP localization. An incomplete but significant increase in nuclear GFP localization relative to the control (no NLS, just GFP) was observed using Leica image analysis software (LASX 2.0, Leica). Nevertheless, a large amount of GFP still remained in the cytosol when using SV40 NLS. Current work is comparing the efficiency of SV40 NLS mediated nuclear localization of GFP in fish cell lines to that mediated by an endogenous tilapia NLS. We have cloned an endogenous tilapia NLS and are currently testing whether this endogenous NLS improves nuclear localization of ectopic GFP in tilapia OmB cells. Optimization of nuclear localization of Cas9 will aid future gene editing studies in the OmB cell line and in cells from related fish species.Support or Funding InformationThis study was funded by a grant from NSF (IOS‐1656371).
Lysosome‐MVB Encountering and Inflammatory Exosome Release Controlled by Acid Sphingomyelinase in Podocytes during Hyperhomocysteinemia
Authors: Li Guangbi , Huang Dandan , Camus Sarah , Zou Yao , Ritter Joseph K. , Li Pin-Lan 
Published: April 2020

Previous studies have demonstrated that overexpression of acid sphingomyelinase (ASM) enhances NLRP3 inflammasome activation and podocyte‐derived inflammatory exosome release during hyperhomocysteinemia (hHcy). However, the mechanism underlying the enhancement of inflammatory exosome release remains unclear. The present study tested a hypothesis that overexpression of ASM blocks lysosome‐MVB encountering via inhibition of lysosome trafficking, leading to enhanced inflammatory exosome release from podocytes during hHcy. Nanoparticle tracking analysis showed a largely increased urinary exosomes in WT/WT mice with hHcy, but not in Smpd1−/− (ASM gene knockout) mice. In Smpd1trg/Podocre (podocyte‐specific ASM gene overexpression) mice, this increase in urinary exosomes was not only during hHcy, but also on the ND. Increased exosomes were podocytes‐derived by detection of podocin as podocyte marker and CD63 as exosome marker. Biochemical analyses showed that NLRP3 products, IL‐1β and IL‐18 significantly increased in hHcy in WT/WT and Smpd1trg/Podocre mice, but not in Smpd1−/− mice. In vitro, we found that Hcy stimulation enhanced release of exosomes out of podocytes from WT/WT and Smpd1trg/Podocre mice, but it had no effect on exosome release from podocytes of Smpd1−/− mice. Exosomes were isolated with ultracentrifugation and purified with exosome purification kit from podocyte culture medium. It was found that Hcy increased CD63 in purified exosomes from WT/WT and Smpd1trg/Podocre podocytes, but it had no effect in Smpd1−/− podocytes. ELISA analysis showed that these increased exosomes contain a lot of IL‐1β and IL‐18 when podocytes were stimulated by Hcy. Super‐resolution microscopy showed the interaction of lysosomes with MVBs. Lysosomes encountered and fused to MVBs much less in podocytes from WT/WT and Smpd1trg/Podocre mice when they were treated with Hcy. In Smpd1−/− podocytes, Hcy failed to change the lysosome‐MVB encounters and interactions. ML‐SA1 as a cell‐permeable lysosome trafficking stimulator via activation of lysosomal TRPML1 channel markedly increased lysosomes movement toward nucleus. Hcy significantly reduced lysosomes movements to nucleus and overexpression of Smpd1 gene enhanced the effect of Hcy. In Smpd1−/− podocytes, ML‐SA1‐induced lysosome movement toward nucleus remained unchanged during Hcy treatment. These results suggest that ASM‐mediated sphingolipid signaling controls lysosome function determining inflammatory exosome release from podocytes during hHcy.Support or Funding InformationThis study was supported by NIH grants DK54927 and DK102539.
Four weeks of environmentally relevant second hand smoke exposure decreased neuronal excitability of cardiac vagal neurons in the dorsal motor nucleus of vagus
Authors: Pan Shi-Yue , Sun Jun-Qing , Chen Chao 
Published: April 2020

BACKGROUNDSecond hand smoke (SHS), a main indoor air pollutant, is a significant risk factor for cardiovascular morbidity and mortality. We previously showed that exposure to SHS, for as short as four weeks, significantly reduced heart rate variability (HRV), suggesting a decreased vagal regulation of the heart. We further showed that, in cardiac vagal neurons in the nucleus ambiguus (NA), SHS exposure significantly reduced spiking response to depolarizing current injections and increased current‐ and voltage‐threshold for action potential generation. However, we have no information on the effects of SHS exposure on cardiac vagal neurons in the dorsal motor nucleus of vagus (DMNV). Here, we sought to test the hypothesis that SHS exposure decreases neuronal excitability of cardiac vagal neurons in the DMNV.METHODSSeven‐week old male C57BL/6J mice underwent surgeries to retrograde label cardiac vagal neurons by applying a fluorescent dye (DiI) to the sinoatrial node. Two weeks after surgery, mice were randomly assigned to either filtered air‐ (FA, n = 14) or SHS‐exposed (n = 16) group. The SHS exposure (6 hr/d, 5 d/wk) was set at a level that is similar to that of a smoky bar (3 mg/m3). After four weeks of FA or SHS exposure, whole‐cell current clamp recordings in brainstem slices were performed on anatomically identified cardiac vagal neurons in the DMNV with fluorescent labeling.RESULTSDMNV cardiac vagal neurons from the SHS‐exposed group were significantly more hyperpolarized than the FA control group (−53.3 ± 0.9 mV vs. −55.7 ± 0.7 mV, FA vs. SHS respectively, p < 0.05). Four weeks of SHS exposure also significantly decreased neuronal spontaneous spiking activity at a holding potential of −45 mV (1.6 ± 0.4 Hz vs. 0.8 ± 0.2 Hz, FA vs. SHS respectively, p < 0.05). The decreased spontaneous activity was not associated with the spiking response to depolarizing current injections from a holding potential of −60 mV, current and voltage threshold for action potential generation, action potential peak and afterhyperpolarization. Furthermore, there was no significant difference in the neuronal input‐output relationship of DMNV cardiac vagal neurons between FA control and SHS‐exposed groups, suggesting an unchanged dynamic responsiveness to stimuli.CONCLUSIONSFour weeks exposure to environmentally relevant level of SHS decreased neuronal excitability of cardiac vagal neurons in a region‐dependent manner – reduced resting membrane potential and spontaneous spiking activity in the DMNV and reduced spiking response to depolarizing current injections and increased threshold for action potential generation in the NA.Support or Funding InformationFunding: R01 ES025229
Skin and Kidney Contribute to Salt‐Sensitive Hypertension in the SAME rat
Authors: Mullins Linda Jane , Ivy Jessica , Rakova Natalia , Mueller Dominik , Mullins John 
Published: April 2020

Loss of Hsd11b2 causes the syndrome of apparent mineralocorticoid excess (SAME) and salt‐sensitive hypertension, which has been demonstrated in the SAME rat
1. We aimed to determine how salt‐sensitive hypertension developed in young SAME rats.Hsd11b2−/− rats were hypertensive (158 versus 119mmHg controls; measured under anaesthesia) and plasma volume contracted (haematocrit 0.49 versus 0.42 control) by 5 weeks of age and showed profound hypokalemia (2mM plasma K+ versus 4.5mM control) the latter being evident from 15 days of age.Adult kidney RNA‐seq analysis suggested a coordinate down‐regulation of electrolyte and water transporters, while a‐ENaC (Scnn1a; 1.66‐fold), the flow‐induced BK channel (Kcnma1; 2.65‐fold) and the HKA2 channel (Atp12a; 4.29‐fold) were significantly up‐regulated. Young Hsd11b2−/− animals exhibited polydipsia and polyuria from weaning age (~20 days old) and, as with adults, they excreted a higher proportion of ingested Na+ (56%) through the kidney, compared to controls (38%), suggesting that the coordinate changes in nephron transporters are already in place around weaning age.Notably, we observed increased skin electrolytes in neonatal animals, (by drying and ashing pelts and determining electrolytes using atomic absorption spectroscopy). Neonatal Hsd11b2−/− animals retained higher [Na+] in their pelts than the controls.Our data suggest that increased Na+ intake [from mothers’ milk] results in increased Na+ storage in skin at the expense of K+. The ensuing hypokalemia primes the developing nephron to deliver increased Na+ and water to the connecting tubule and cortical collecting duct by weaning age. Glucocorticoid‐induced Na+ uptake in the kidney, and reduced Na+ storage capacity in the pelt, both then contribute to progressive salt‐sensitive hypertension.Support or Funding InformationBritish Heart Foundation Centre of Research Excellence Award (RE/08/001/23904); Kidney Research UK project grant
Investigating the Protein Kinase C Polarized Growth‐Related Protein Complex in Aspergillus nidulans
Authors: Jackson-Hayes Loretta , Craft Alexis , Hameed Muhammad , Hines Zariah , Freyaldenhoven W. Toler , Hill Terry 
Published: April 2020

The long‐term goal of our research is to advance understanding of the mechanisms involved in hyphal growth, as well as maintenance of cell walls in filamentous fungi. Recently our work has focused on proteins that localize to sites of cell wall synthesis, namely growing cell apices (hyphal tips) and forming septa. This project focusses on Protein kinase C (PkcA). In previous work, we identified a mutation in the gene that encodes an orthologue of protein kinase C in our model organism Aspergillus nidulans, which results in reduced cell wall integrity. We showed that wild type PkcA localizes to growing hyphal apices and the contractile actomyosin ring at forming septa, and we identified amino acid sequences within PkcA that are required for PkcA to localize to these sites of cell wall synthesis. This project seeks to uncover the protein‐protein interactions involving PkcA and determine the PkcA‐complexed proteins it phosphorylates and those that it does not. Previously we reported that PkcA forms a complex with the formin SepA, and that this interaction requires the FH2 domain of SepA. Published results from another group suggested that the Rho‐type GTPase Rho4 plays a role in SepA function and that Bud3 acts as a GEF for Rho4. Here we show that the putative Rho binding domain of PkcA (HR1A) is sufficient for its interaction with SepA. Fluorescence microscopy of a dually labeled A. nidulans strain illustrate that although PkcA‐mRFP and Rho4‐GFP often reside at the contractile actomyosin concurrently, PkcA precedes Rho4 in ring constriction and yeast two‐hybrid assays failed to show an interaction between PkcA and Rho4. Further, neither of 4 other Rho homologues tested interacted with PkcA in yeast two‐hybrid experiments. Sequence analysis of the FH2 domain of SepA revealed a Rho‐like motif encompassing amino acids 377–408 having 43.3% sequence identity with RhoA. This result suggests that the sufficiency of the HR1A domain for PkcA’s interaction with SepA is due to its affinity for the Rho‐like motif within FH2. Co‐immunoprecipitation of GFP‐PkcA followed by tandem mass spectrometry and proteomics analysis of complexed proteins identified chitin synthase B and the glucan synthase FksA within the PkcA complex. Ongoing studies will seek to determine if these proteins are substrates for PkcA.Support or Funding InformationNSF RUI 1615192