bluuebunny/crossref_metadata_2025_split huggingface dataset:
Competitive Counterflow (CCF) To Distinguish Between Transported And Non‐Transported Inhibitors Of MATE1 and MATE2‐K
Authors: Martinez-Guerrero Lucy J. , Wright Stephen H. 
Published: April 2020

The MATEs (hMATE1 & hMATE‐2K) are membrane proteins expressed in the apical membrane of renal proximal tubule cells that mediate the flux of organic cations into the filtrate. In this study we set to test CCF as a method to identify substrates of pharmacological interest for both MATEs.We first determined IC50 values for inhibition of MPP transport into CHO cells that stably expressed either MATE1 or MATE2‐K for several clinically important compounds: Amlodipine (IC50= 7‐6 μM), Atenolol (10‐4 μM), Dofetilidae (4‐0.8 μM), Entecavir (302‐140 μM), Lamivudine (336‐53 μM), Omeprazole (53‐8 μM), Propranolol, (16‐5 μM) and Trimethoprim (6‐0.4 μM). For CCF, cells were exposed to ~12 nM [3H]MPP for 40 min, sufficient to reach steady state. At time 0, the medium was removed and replaced with medium containing either ~12 nM [3H]MPP (control) or ~12 nM [3H]MPP plus one of the test compounds, or a known MATE substrate (e.g., Metformin); PAH (1mM) was used as a known non‐transported compound. After an additional 5 min incubation, radiolabeled MPP remaining in the cells was determined. If the extracellular test compound was transported, the associated turnover of the transporter should result in net loss of labeled MPP from the cells. Each test was performed (in sextuplicate) in 2–3 separate experiments.Whereas exposure to 1mM PAH resulted in no loss of MPP from the cells (which was distributed to steady state), exposure to metformin resulted in net loss of radiolabeled substrate (70%). Of the test compounds, Atenolol, Amlodipine & Lamivudine supported a net loss of [3H]MPP (70‐30%) from both MATE1 & MATE2K‐expressing cells, whereas Entecavir reduced total accumulation of MPP only for MATE2K expressing cells. There was no loss (or gain) of MPP induced by Dofetilidae, Propranolol or Omeprazole, suggesting they are non‐transported inhibitors for both MATEs.These data support the conclusion that the method of CCF is applicable for identification of MATE substrates, and identified a difference in the selectivity profile of hMATE1 & hMATE2K.Support or Funding InformationSupported by NIH Award 5R01GM129777
The effects of varying DNA flap length from 20 to 50 deoxynucleotides on the mechanism of single‐strand annealing in Saccharomyces cerevisiae
Authors: Odango Jane , Fregoso Fred , Camberos Juan , Fischhaber Paula 
Published: April 2020

Mutagens constantly threaten the integrity of genetic material by causing damage in the form of double‐strand breaks (DSBs). Such damage is typically repaired through various modes of homologous recombination (HR). This project focuses on a form of HR known as single‐strand annealing (SSA), which is triggered when a DSB is located between two DNA repeats. This pathway generates 3' overhanging single‐stranded DNA “flaps” which are cleaved by the Rad1‐Rad10 endonuclease, aided by the mediator protein Saw1 in S. cerevisiae. Recent literature has demonstrated that Saw1‐dependent recruitment of Rad1‐Rad10 to damage sites, and SSA repair proficiency, are both a function of flap length. In this study, we set out to elucidate the precise flap length threshold requiring Saw1 for optimal SSA repair in vivo through fluorescence microscopy and real‐time quantitative PCR (qPCR).In order to investigate the importance of DNA flap length on Rad1‐Rad10 and Saw1 recruitment, we designed yeast strains generating 20, 30, or 50 nt flaps during SSA. A DSB was induced at a fluorescently labeled site in the yeast genome (DSB‐RFP), allowing us to monitor the recruitment of Rad10‐YFP via fluorescence microscopy. The percentages of cells containing colocalized foci in strains either wild‐type or mutant in Saw1 were quantified, revealing that Saw1‐mediated recruitment of the Rad1‐Rad10 complex increased with flap length. Surprisingly, we found that Saw1 was actively recruited to the DSB site across all flap length substrates via experiments with a Saw1‐CFP strain. Analyses via qPCR were also carried out to quantify SSA repair products formed in WT and saw1Δstrains. The data show diminished repair product formation in the absence of Saw1 as flap lengths increased. Together, these results suggest that Saw1 is required for Rad1‐Rad10 recruitment and efficient SSA repair in longer flap substrates with ~20–30 nt representing the key size triggering Saw1‐dependence for repair. Current efforts are underway to determine the Saw1‐dependence of proficient SSA repair under conditions in which background induction is not amplified by qPCR.Support or Funding InformationThe authors thank our Department and the NIH SC3 program for funding.
The EBP50 and VDAC1 Interaction Mediates Cytoprotective Signaling on Mitochondria during Oxidative Stress
Authors: Jou Tzuu-Shuh , Chang Pei-Yu 
Published: April 2020

Mitochondria, the organelles controlling ATP production and cellular metabolism, play a vital role in a number of cellular stress responses by communicating with other cellular compartments, for example, mitochondria‐associated membranes (MAMs), the tightly membrane contact site between mitochondria and endoplasmic reticulum (ER). We recently found a scaffold protein, Ezrin/radixin/moesin (ERM)‐binding phosphoprotein 50 protein (EBP50), plays a cytoprotective role on mitochondriam. Knockdown of EBP50 elicited severe mitochondrial morphological change fro fusion to fission and augmented apoptotic rate upon H2O2‐induced stress and could be rescued by overexpressing siRNA resistant construct of EBP50 in Madin‐Darby Canine Kidney (MDCK) cells. Furthermore, PDZ1 domain of EBP50 interacted with one of pivotal MAMs proteins, voltage‐dependent anion channel 1 (VDAC1), which is localized on outer mitochondrial membrane (OMM). The N‐terminus of VDAC1 regulates its oligomerization and channel activity. Amine‐terminal deletion (aa 1‐26) of VDAC1 diminished EBP50‐VDAC1 interaction while carboxyl‐terminal deletion (aa 240–283) retained the intact association. Moreover, the central component of the translocase of outer membrane (TOM) Tom20 also participated in EBP50‐VDAC1 interaction.Our study paves the way for unraveling the detailed mechanisms and provides molecular insights into how mitochondria are protected from damaging during oxidative stress response.
Controlled Cortical Impact Increases the Relative Abundance of Chondroitin 6‐Sulfate in the Lateral Posterior Thalamus
Authors: Logsdon Aric , Alonge Kimberly , Wight Thomas , Guttman Miklos , Banks William 
Published: April 2020

The brain is limited in its capacity to repair and reorganize neural networks following traumatic brain injury (TBI). Sulfated chondroitin sulfate (CS)‐glycosaminoglycan (GAG) extracellular matrix chains are known to influence formation and maintenance of neural networks, and maladaptive changes to the CS‐GAG abundance or sulfation pattern are associated with neurocircuit dysfunction. Recent reports indicate that TBI can alter the sulfation pattern of CS‐GAG chains, however, little is known about the anatomical and functional relevance of these changes. In this study, adult male C57BL/6 mice were subjected to a TBI via a controlled cortical impact (CCI) to the somatosensory cortex (SSC), where an established thalamocoritcal connection between the SSC and the lateral posterior thalamus (LPT) exists for sensorimotor function. At 7 and 28 days following CCI, immunofluorescence analysis of the ipsilateral, but not the contralateral LPT revealed a significant increase in the 6S‐CS sulfated GAG marker CS‐56. To analyze the relative changes in all CS‐GAG sulfation, CS disaccharides were released from both ipsilateral and contralateral SSC and LPT regions and quantified using a recently optimized LC‐MS/MS + MRM technique, which can differentiate between the relative abundance of five specific CS isomers (Δ4S‐, Δ6S‐, Δ4S6S‐, Δ2S6S‐, Δ0S‐CS) and dermatan sulfate (Δ2S4S‐DS). Here, we show that both the ipsilateral SSC and LPT exhibited higher percentages of Δ6S‐CS disaccharides compared to the contralateral brain regions, as well as the sham control brain regions. To determine whether the increase in 6S‐CS abundance in the SSC and LPT associate with sensorimotor function, CCI mice were subjected to the rotarod task to measure sensorimotor performance. Mice exposed to CCI exhibited a decrease in their latency to fall from the rotarod, which inversely correlated to the percentage of Δ6S‐CS. Collectively, these data suggest that CCI alters the CS‐GAG sulfation pattern within the thalamocortical circuitry, and these changes may contribute to persistent sensorimotor dysfunction. Future studies will investigate novel therapeutic interventions targeting CS‐GAG chains to potentially improve outcomes from TBI.Support or Funding InformationR01GM127579 to M.G.R01AG046619 to W.B.F32DK12266201 to K.A.
Exploring the Effects of a V104F/V477F Double Mutation on CYP2B6 Activity Towards Efavirenz
Authors: Prewett Alisa , Riegsecker Ben , Angle Ethan , Cox Philip 
Published: April 2020

BackgroundThe Cytochromes P450 2B6 (CYP2B6) and 2A6 (CYP2A6) participate in the metabolism of the anti‐HIV drug Efavirenz (EFV). CYP2B6 monohydroxylation of EFV results in the formation of 8‐hydroxyefavirenz (8‐OHEFV) whereas CYP2A6 monohydroxylation of EFV forms 7‐hydroxyefavirenz (7‐OHEFV). To date, little is known about the structural characteristics of these enzymes that lead to differential EFV metabolite formation. In order to better understand such characteristics, we sought to mutate CYP2B6 towards CYP2A6 in hopes of uncovering amino acids critical for the formation of 8‐OHEFV. Specifically, we decided to focus on two phenylalanine residues in the CYP2A6 active site that are valines at the analogous positions in CYP2B6.MethodsThe valine residues at positions 104 and 477 of CYP2B6 were mutated to phenylalanines using site directed mutagenesis, creating a V104F/V477F double mutant. Wild type (WT) and mutant CYP2B6 expression was accomplished in COS7 cells and, following treatment with EFV, metabolites were extracted and detected using a novel gas chromatography‐mass spectrometry (GC‐MS) method after chemical derivatization using BSTFA + 10% TMCS. Protein expression was assessed via western blotting.ResultsWe observed little to no difference in the protein expression of WT and mutant CYP2B6 proteins. 8‐OHEFV production in the COS7 system was detected for wild type and mutant CYP2B6, but no 7‐OHEFV production was observed. Interestingly, GC‐MS analysis of 8‐OHEFV formation by the V104F/V477F mutant indicates a marked increase in 8‐OHEFV production, suggesting the combination of these mutations might enhance 8‐OHEFV formation by CYP2B6.Future GoalsFuture experiments will seek to characterize each single mutant and determine kinetic parameters for 8‐OHEFV formation by all three mutant CYP2B6 proteins.Support or Funding InformationFunding for this work was generously provided by the Department of Biology and Chemistry at Azusa Pacific University and the Faculty Research Council of Azusa Pacific University
Changes in the gut microbiota of wild mice according to environment
Authors: Cho Seongbeom , Kim Junhyung , Guk Jae-Ho , Song Hyokeun , Suh Jun Gyo , Seong Je Kyung 
Published: April 2020

BackgroundThe gut microbiota is closely linked with the health and disease status of hosts. This could be affected by multifactorial factors, among which environmental factors have a great effect on the gut microbiota, which is the cause of different phenotypes in mice. Here, we investigated the changes in the gut microbiota of wild mice according to habitat and diet.Methods and ResultsForty‐nine Mus musculus were captured from fields that were separated by rivers, roads, and mountains in Korea. Sixteen representative wild mice were transferred to laboratory animal facilities and their gut microbiota was analyzed at 2–4 weeks intervals up to 20 weeks. At the phylum level, 16 phyla were identified; among them, four phyla (Firmicutes, Bacteroidetes, Epsilonbacteraeota, and Proteobacteria) comprised the core gut microbiota and accounted for about 98% of the total microbiota. In alpha diversity, the number of observed OTUs and richness significantly reduced, while the evenness significantly increased. In beta diversity, the distribution of the gut microbiota initially differed depending on the habitat of wild mice. However, as wild mice adapted to laboratory animal facilities, the gut microbiota clustered together in the PCoA plot and the volatility of beta diversity also showed distinct differences. Furthermore, the relative abundance of Staphylococcus (p < 0.01) and Alistipes (p < 0.05) tended to increase at the genus level.ConclusionsDue to the variety of wild environments in which wild mice are exposed, wild mice have a greater diversity and distribution of the gut microbiota than mice exposed to constant environments. As wild mice adapted to a constant environment, the gut microbial diversity decreased and the distribution of the gut microbiota became constant. This study provides a new approach, gut microbiota analysis, in the assessment of environmental exposure in wild mice, which is limited based on phenotyping and genotyping.Support or Funding InformationThis research was supported by the National Research Foundation of Korea (NRF‐2018R1A2B6002396) and the Korea Mouse Phenotyping Project (2016M3A9D5A01952417) of the Ministry of Science, ICT and Future Planning.
Genetic Association of ACE Polymorphism with Sarcopenia and Effects of Genetic Regulation of ACE Activity on Muscle Function
Authors: Kim Chulhyun , Kim Hyeoijin , Song Keon-Hyung , Kwon Insu , Ambegaonkar Jatin 
Published: April 2020

The purpose of this study was to investigate associations between skeletal muscle mass and ACE polymorphism, or sarcopenia‐related candidate gene, and to examine the effects of genetical angiotensin‐converting enzyme (ACE) activity on physical performance and respiratory muscle function in Korean adults. For this study, 1,464 Korean adults aged 18~79 were recruited. The anthropometrics were 1.69±0.06m (height), 63.1±5.5kg (weight), 29.4±6.5% (percent body fat), and 24.5±3.0kg/m2 (body mass index). Fat‐free mass, physical performance, gait speed (Timed Up‐and‐Go), pulmonary function (maximal inspiration and expiration pressure) and fatiguing maximum voluntary isometric contraction (40% MVIC) of ankle plantar flexor muscles with dynamic Electromyography were tested. Data of body composition and muscle functions were analyzed using one‐way ANOVA and post‐hoc Scheffe test for continuos variables and the Chi‐square test and host‐hoc Bonfferioni correction for countable variables. Blood samples for the whole blood and serum were obtained and assayed for circulating ACE activity and the ACE I/D genotype. The distribution of ACE polymorphism throughout the groups was analyzed using the Chi‐square test and host‐hoc Bonferroni correction for genetic distribution and multiple logistic regression analysis for the odds ratios (ORs) with 95% confidence intervals (CIs). We found that the distribution of ACE Indel genotype for sarcopenia was significantly different from normal population who have normal or high fat‐free mass index. Especially, we found a significantly higher (p<.05) distribution of DD genotype in sarcopenia participants (27%) than the incidence (12.5%) in normal population. The D allele of ACE genotypes in older adults indicated significantly slower gait speed than I allele of them in the Timed Up‐and‐go test (p<.05), and significantly lower levels for maximal inspiration and expiration pressure (p<.05). The II genotype in older adults indicate the highest tolerance of muscle fatigue during muscle contraction (p<.05). In conclusion, ACE Indel polymorphism and level of ACE activity is associated with sarcopenia in older adults and would genetically regulate muscle function in part. Our findings suggest that genetical concentrations of angiotensin‐converting enzyme should play a role in determining low muscle mass, slow walking speed, respiratory muscle function, and fatigue after muscle contraction in individuals. Therefore, examining ACE Indel polymorphism and ACE activity level could be used to predict sarcopenia and muscle function, and subsequently this information can be applicable to prevention and treatment to the aging muscle.Support or Funding InformationThis 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 (358‐2011‐1‐G00025).
Effects of Artesunate with Paclitaxel on the Proliferation and Morphology of Androgen‐sensitive (LNCaP), Androgen‐insensitive (PC‐3) Human Prostate Cancer Cell Lines and Normal Epithelial Prostate cell line (RWPE‐1)
Authors: Adedeji Dolapo , Fabian Juan , Adedeji Adetayo , Payne Gloria 
Published: April 2020

BackgroundProstate cancer is one of the diseases worldwide that causes cancer‐related deaths in men and is the second most common cancer affecting thousands of men each year in the United States behind only lung cancer. Artesunate (ART) is part of the artemisinin drugs for the treatment of malaria. Paclitaxel (PTX) is an anticancer agent that belongs to the Taxane class and is used for the treatment of metastatic hormone‐refractory prostate cancer.PurposeThe aim of this study was to examine the effects of combining ART with PTX on human prostate cancer cell lines ‐ LnCAP and PC‐3, normal epithelial prostate cell line RWPE‐1 and observe any morphological change using Scanning Electron Microscope (SEM).MethodIn vitro anti‐proliferative 3‐(4,5‐Dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS) assay was used to assess the effects of ART with PTX on human prostate cancer cell line (LnCaP and PC‐3) and normal prostate cell line (RWPE‐1) after 72‐ and 120 – hour exposure at a concentration range from 1 nm – 100 μM. For the SEM images, cell lines were cultured with thin films of gold, titanium and zinc oxide deposited by magnetron sputtering on silicon substrate at room temperature. Images of samples with and without the drugs were acquired.ResultsThe average cell death observed for both LnCAP and PC‐3 cell lines was 40% and 73% after 72‐ and 120‐ hours incubation with ART and PTX at different combination ratios of 1:1; 1:2 and 2:1 for ART: PTX and 1:1; 1:2 and 2:1 for PTX: ART with concentrations ranging from 1 nm – 100 μM. For normal epithelial prostate cell line, RWPE‐1, the average cell death observed ranged from 0.35% – 23% using the same combination ratios. Cell adhesion was detected on silicon substrates and on thin films of gold, titanium and zinc oxide. Morphological changes were observed when samples were treated with and without drugs. These SEM images were associated with cell lines undergoing apoptosis when compared with control (images with no drugs).ConclusionAlthough further observations are needed, it appeared that combination of ART with PTX displayed cytotoxicity regardless of whether the prostate cancer cell line is androgen‐sensitive or androgen‐insensitive after 72 and 120 ‐ hour exposure with little or no effect on normal epithelial prostate cell line. Therefore, combination of both drugs may aid in reducing cardio‐toxicity exerted by PTX alone thereby reducing the prostate cancer mortality rate in men.Support or Funding InformationWork support by ECSU Mini‐grant and in part by NSF Award# 1818774
BACTERIAL CELLULOSE MEMBRANES WITH PLASMA‐MODIFIED SURFACES FOR TISSUE ENGINEERING APPLICATIONS
Authors: Benevenuto Luíz Guilherme Dercore , Cruz Sandra Andrea , Alves Eduardo Donato , Achcar Jorge Alberto , da Silva Barud Hernane , Montrezor Luís Henrique 
Published: April 2020

Bacterial cellulose (BC) is a promising biomaterial notable for its features such as strength, hydrophilicity, and biocompatibility, which enable it to be used as implants and scaffolds in tissue engineering. However, its topography does not present the ideal characteristics for cell attachment and development, so surface modifications are required in order to improve the cellular response. The objective of this study was to analyze the effects of oxygen plasma treatment applied to bacterial cellulose (BC) for 2.5 and 5 minutes, together with its effects on cell behavior during 21 days of culture. Osteo‐1 cells were cultured in the absence and presence of BC, at an initial density (day 0) of 1000 cells per 200 μL of osteogenic medium (DMEM + FBS + ascorbic acid + dexamethasone + β‐glycerophosphate), in 48‐well culture plates, for 7, 14, and 21 days, with exchange of the medium every 72 h. Cell counting was performed with a Bio‐Rad TC20™ cell counter, viability was analyzed using the MTT test, and mineralization was quantified by the Alizarin Red test. Alkaline phosphatase (AP) in the culture medium was measured using a commercial LabTest kit. Statistical analysis of the data was performed using ANOVA and Fisher’s test for multiple comparisons. Alkaline phosphatase results: Higher AP was observed for the group with application of the plasma to the BC for 5 minutes, while no statistically significant differences were found among the groups without treatment, treatment for 2.5 minutes, or in the absence of BC, as well as between the days of culture. Culture time effects: Higher cell counts were obtained for the control group without BC and the group with untreated BC. The highest cell counts were observed at 14 and 21 days of culture, with no statistically significant differences among the means. Mineralization and cell viability: The highest responses for mineralization and viability were observed at 7 days of culture, followed by decreases at 14 and 21 days. The results were indicative of changes on the bacterial cellulose surface caused by the oxygen plasma treatment, which influenced the cell features. The findings indicated that plasma treatment for up to 5 minutes changed the topography of the surface, leading to better conditions for cell differentiation and development from Osteo‐1 to osteoblast, as indicated by the higher AP concentration for this group. The treatment for 5 minutes led to enhanced cellular responses on the bacterial cellulose, contributing to better cell development.Support or Funding InformationFundingFAPESP (grant #2018/14840‐4).