bluuebunny/crossref_metadata_2025_split huggingface dataset:
Optimized Induction of Neurospheres from Porcine Mesenchymal Stem Cells
Authors: Schomberg Dominic T. , Wood Aleksandar , Singh Harpreet , Meudt Jennifer J. , Shanmuganayagam Dhanansayan 
Published: June 2021

Neurospheres are free‐floating clusters of cells that provide researchers a unique method to investigate neural stem cells. Stem cells typically grow as an adherent monolayer on the culture surface, however, when introduced to proper media and growth factors without adherent substrates, these stem cells will form non‐adherent three‐dimensional aggregates called neurospheres. Induction of neurospheres increases multipotency and regenerative capacity of some stem cells, such as adipose‐derived mesenchymal stem cells (adMSCs). Currently, there are no standardized methods for neurosphere induction and evaluation which limits their utility. Studies into cancer, neural development, and regenerative medicine would all benefit from an optimized neurosphere induction protocol. We aimed to establish an optimized protocol for inducing neurospheres from adMSCs in an efficient and reproducible manner.Primary swine adMSCs were induced to neurospheres with media comprised of varying concentrations of neurobasal medium, B27, and glutamine, epithelial growth factor (EGF) and basic fibroblast growth factor (bFGF) and morphometric parameters of growth (total neurospheres, total cells, cell density and neurosphere diameter) were evaluated daily for eight days. After the eight‐day induction period, the multipotency of neurospheres was determined by assessing presence of neuronal markers nestin, Sox2 and Oct4. We also evaluated for ideal conditions under which neurospheres formed without any signs necrosis at the core. Our optimized neurosphere culture methods produce neural stem cell that best maintain neural‐multipotency and provide a standardized and reproducible protocol for neuronal stem cell propagation.Support or Funding InformationThe research was supported by the Biomedical & Genomic Research Group Discretionary Fund (University of Wisconsin‐Madison), NF North Central, NF Network, and NF Team Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the function of the histone methyltransferase PRMT1 during post‐embryonic development in Xenopus tropicalis.
Authors: Shibata Yuki , Shi Yun‐Bo 
Published: June 2021

Anuran amphibians such as Xenopus tropicalis offer a unique opportunity to study how the adult organs and their organ‐specific stem cells are formed in vertebrates during the so‐called postembryonic development. The postembryonic events such as the maturation/remodeling of many organs are totally regulated by thyroid hormone (TH). Of particularly interest among TH target genes is protein arginine methyltransferase1 (PRMT1), which can function as a TR co‐activator. We have shown that PRMT1 mRNA and protein expression peaks at the climax of metamorphosis in the intestine when adult stem cells are forming de novo and proliferating, and PRMT1 overexpression enhanced the expression of TH target genes and increased the number of adult intestinal stem cells during metamorphosis, suggesting a critical role during the formation of adult stem cells in the intestine. To determine the function of the endogenous PRMT1 gene during post‐embryonic development and intestinal stem cell development, we adapted the gene editing technology to knockout PRMT1 gene in X. tropicalis.We used Transcriptional activator–like effector nuclease (TALEN) technology to generate PRMT1 knockdown frogs (F0). After generating PRMT1 heterozygous frogs (F1) with out of frame mutations from the F0 animals, we tried to produce PRMT1 homozygous knockout frogs by intercross the F1 frogs. We measured the survival rate, animal size, and the developmental stage of the resulting tadpoles, including wild type, heterozygous and homozygous animals.As the result, we successfully generated PRMT1 heterozygous (F1) frogs including out frame mutation (8 base deletion), and PRMT1 homozygous knockout (F2) animals by intercross with F1 frogs. Surprisingly, all PRMT1 homozygous tadpoles died within 14 days after fertilization although they completed embryogenesis to form apparently normal tadpoles. In addition, morphological analysis showed that PRMT1 knockout tadpoles delayed animal development and growth.PRMT1 knockout tadpoles completed embryogenesis normally but had delayed development and tadpole lethality. The availability of knockout tadpoles will allow us to study the role of PRMT1 during T3 ‐induced metamorphosis, thus overcoming the difficulty to study PRMT1 during postembryonic development in mammals due to the lethal phenotype in PRMT1 knockout mice.Support or Funding InformationJSPS Research Fellowship for Japanese Biomedical and Behavioral Researchers at NIHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Muscle specific MnSOD deficiency leads to complex II‐specific inactivity of ETC and contractile dysfunction, but increases muscle mass
Authors: Ahn Bumsoo , Ranjit Rojina , Pharaoh Gavin , Piekarz Katarzyna , Matsuzaki Satoshi , Premkumar Pavithra , Riddle Kaitlyn , Humphries Kenneth , Richardson Arlan , Van Remmen Holly 
Published: June 2021

Multiple pathological conditions exhibit excess mitochondrial ROS and muscle weakness in parallel, including aging, denervation and cancer cachexia. To directly test the role of mitochondrial ROS in skeletal muscle weakness and atrophy we generated a skeletal muscle specific knockout mouse model in which MnSOD (Sod2), the primary superoxide anion scavenger in the mitochondrial matrix, was deleted using a Cre‐Lox approach driven by a constitutively active human skeletal actin promoter (HSA‐Cre). Female wild type and MnSOD deficient (mSod2 KO) mice were studied at 6–8 months of age. In mSod2 KO mice, the observed rate of superoxide generation from isolated skeletal muscle mitochondria was significantly elevated, and hydrogen peroxide generation using permeabilized fibers increased by ~5 fold. ATP generation rate and electron transport chain (ETC) activity were severely reduced in response to Complex II linked substrate, suggesting superoxide‐induced complex II inactivity. Reduced ATP generation rate and ETC activity were consistent with severe exercise intolerance in mSod2 KO mice. To determine the effects of mitochondrial ROS on skeletal muscle weakness, we first tested in situ contractile function using gastrocnemius. Maximum isometric specific force was decreased by ~30 % in mSod2 KO mice with direct stimulation on muscle, but the reduction in force was further diminished (~50%) after stimulation of the sciatic nerve suggesting neuromuscular disruption. Morphological assessment exhibited increases in NMJ size and fragmentation in mSod2 KO mice supporting that mitochondrial dysfunction can feed back to initiate NMJ disruption. Surprisingly, gastrocnemius muscle mass was increased in mSod2 KO mice, which was associated with increased numbers of central nuclei and hyperplasia via fiber branching. In summary, our data suggest that excess mitochondrial superoxide caused a selective loss of complex II activity and mitochondrial defects, leading to severe exercise intolerance. Increased cytoplasmic hydrogen peroxide induced necrosis‐mediated regeneration, which increased muscle mass by fiber branching.Support or Funding InformationNIH/NIA to Dr. Van Remmen (R01 AG050676) and a VA Merit grant to HVRThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modeling Calcium‐Induced Inhibition of Oxidative Phosphorylation
Authors: Malyala Sathyavani , Zhang Yizhu , Bazil Jason N. 
Published: June 2021

Ischemic myocardium is characterized by a chronic elevation in cellular calcium concentration. In this setting, mitochondria accumulate calcium to levels that compromise their ability to synthesize ATP. Although total mitochondrial calcium can reach up to 1 M, the vast majority is presumed to be present as insoluble calcium phosphate granules. We recently reported calcium levels below thresholds that trigger mitochondrial permeability transition significantly lowers the rate of ADP‐stimulated respiration. To explain these data, we tested the following hypotheses using a novel mathematical model of cardiac mitochondrial energetics and calcium handling: i) depressed membrane potential caused by sodium/calcium cycling lowering the driving force for ATP production; ii) direct inhibition of calcium on mitochondrial processes critical for ATP production; iii) calpain activation leading to a decrease in ATP synthesis capacity; iv) depletion/reduction of available ADP for phosphorylation caused by calcium biding, incorporation into calcium phosphate granules, or net loss of adenine nucleotide. The model includes mechanistic descriptions of mitochondrial metabolism and energetics in addition to calcium uptake, sequestration, and efflux pathways. Based on the model analysis, sodium/calcium cycling lowers mitochondrial membrane potential, but does not lead to lower rates of oxidative phosphorylation. However, direct inhibition of calcium on ATP production did agree with these data. We also found that calpain activation is a possible explanation, but we experimentally ruled this mechanism out. Finally, ADP complexation with calcium did not affect the rate of oxidative phosphorylation, but net loss of nucleotides via incorporation into calcium phosphate granules or efflux out of mitochondria can explain these data. The model will be used to design further experiments to determine the most likely mechanism capable of explaining the calcium‐induced inhibition of oxidative phosphorylation.Support or Funding InformationR00‐HL121160This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Protective Effects of Atrial Natriuretic Peptide Infusion in Salt‐Sensitive Hypertension
Authors: Ilatovskaya Daria , Levchenko Vladislav , Zietara Adrian , Winsor Kristen , Spires Denisha R. , Staruschenko Alexander 
Published: June 2021

Atrial Natriuretic Peptide (ANP) is a hormone secreted by cardiomyocytes in response to increased atrial volume; it stimulates water and sodium excretion by the kidneys and relieves pressure on the circulatory system. In our previous studies we compared wild type Dahl Salt Sensitive (SS) rats to SS rats lacking the Nppa gene which encodes ANP (SSNppa−/−). The data revealed that salt‐induced blood pressure development was significantly exacerbated in the SSNppa−/− rats. Furthermore, SSNppa−/− rats exhibited reduced urinary sodium excretion and diuresis, as well as aggravated kidney damage compared to wild type controls (FASEB J 2017 31:855.8.). These data led to a hypothesis that ANP infusion can have beneficial effects during the development of salt‐induced hypertension.Dahl SS rats were fed a high salt diet (HS, 4% NaCl) for 21 days, with a continuous i.v. infusion of ANP (100 ng/kg/day) administered from day 0 or day 14 onward to test preventive and therapeutic effects of ANP, respectively. Blood pressure was monitored throughout the study, and urine samples were collected in metabolic cages on days 0 and 21. At the end of the protocol animals were sacrificed and kidney tissues were harvested and stained with Masson's trichrome to evaluate renal damage. Fibrosis, glomerular injury, and protein casts formation were assessed; urine and plasma samples were also analyzed to estimate electrolyte homeostasis.ANP infusion had beneficial effects on BP and renal function compared to control animals. We observed a decrease in blood pressure in both groups infused with ANP during 0–21 days (21D) and 14–21 days (7D) of a HS challenge. Kidney to body weight ratio did not differ between the groups; however, we observed a trend for reduced renal hypertrophy in the 21D animals. Metabolic cage studies revealed no changes in diuresis; urinary electrolyte analysis showed a more effective sodium excretion in the 21D rats. Assessment of kidney damage demonstrated a decrease in protein casts in both 7D and 21D rats: 3.8% ± 0.47%, 2.7% ± 0.24%, and 2.0% ± 0.41% of the tissue surface in the control, 7D and 21D groups, respectively. Glomerular injury had a similar pattern: scores were 2.86 ± 0.09, 2.62 ± 0.07, and 2.41 ± 0.06. Renal fibrosis was alleviated in the 21D group (1.4% ± 0.18%, compared to 1.9% ± 0.49% and 3.1% ± 0.31% in the 7D and control groups).Therefore, ANP infusion had both preventive and therapeutic effects. Our data revealed that ANP administration during the HS diet challenge resulted in decreased blood pressure, increased electrolyte excretion, and reduced tissue damage.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating Hypotheses through Discovery of Relevant Models of Epithelial Transport
Authors: Sarwar Dewan , Yu Tommy , Atalag Koray , Carlson Brian , Gennari John , Hunter Peter J. , Neal Maxwell , Nickerson David P. 
Published: June 2021

Scientists often utilize computational models to help understand experimental observations or to investigate hypotheses in ways that are not practically achievable experimentally. The first step in constructing such models is to discover existing models which can be reused to help in such an investigation. We are developing novel research tools to enable the discovery and exploration of computational models relevant to specific research questions or hypotheses. This can aid biologists and clinicians to test their clinical or experimental hypotheses from a given collection of disparate mechanisms and/or observations such as diseases, drug actions and clinical observations.Computational tools and standards have evolved over the years such as CellML to encode mathematical models of biological systems; SemGen to annotate models; and the Physiome Model Repository (PMR) to store these models and their comprehensive descriptions to ensure the deposited knowledge is persistent, identifiable, discoverable and sharable. Our long term goal is to develop a knowledge management system capable of constructing multi‐scale renal nephron models from information extracted from PMR to investigate transport phenomena in the kidney at the cellular and subcellular level and the impact on nephron function.We have used the SemGen tool to comprehensively annotate a preliminary cohort of renal epithelial transport models available in PMR to provide the initial knowledgebase for model discovery and exploration, and in particular, to extract a model suitable for testing specific clinical or experimental hypotheses. One use of such exploration is the identification of missing resource(s), which could be addressed via more accurate description of existing resources or the addition of new resource to the repository.Specifically, we present here a web‐based tool enabling scientists to discover and explore epithelial transport models that are relevant to their specific research questions. Biologists and clinicians can use this tool to discover models with specific transport proteins, solute species, genes, anatomical location, or other relevant information. The result of this discovery is a “recipe” describing the computational model that can be assembled to help the user investigate their hypothesis.Support or Funding InformationSupported by the MedTech Centre of Research ExcellenceThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hydrogen Peroxide Increases Intracellular Sodium Concentration in the Medullary Thick Ascending Limb Isolated from Sprague‐Dawley Rats
Authors: Zheleznova Nadezhda N. , Cowley Allen W. 
Published: June 2021

Elevations of renal medullary hydrogen peroxide (H2O2) produce a salt‐sensitive form of hypertension as shown by chronic infusion of H2O2 into the renal medulla of a single remaining kidney of normotensive Sprague‐Dawley (SD) rats (Makino et al., Hypertension, 2003) and salt‐resistant consomic SS.13BN rats. Conversely, chronic medullary infusion of catalase (H2O2 scavenger) into the renal medulla of Dahl salt‐sensitive rats significantly reduced salt induced hypertension (Taylor and Cowley, Am J Physiol Regul Integr Comp Physiol, 2005). The goal of this study was to assess the effects of H2O2 upon intracellular sodium concentration [Na+]i and the role of mTORC1 and mTORC2 signaling in the medullary thick ascending limb (mTAL) isolated from the outer medulla (OM) of SD rats.Methodsrats were anesthetized, the kidney flushed with saline, removed, and mTALs dissected from the inner stripe of the OM at 4°C under a stereomicroscope. The mTALs were attached to poly‐Lysine coated cover slips and incubated with HBSS containing 10 nM of the Na+‐sensitive fluorescence dye NaGreen and 0.05% Pluronic acid F‐127 for 30 min at room temperature. The mTALs were washed and then placed in a heated chamber (37°C) mounted on the stage of an inverted Nikon TE‐2000U microscope for recording.ResultsIt was found that 100 uM H2O2 resulted in a significant increase in [Na+]i (p<0.05). The responses to pretreatment of mTAL with 10 uM PP242 (mTORC1/2 inhibitor), 200 nM rapamycin (mTORC1 specific inhibitor), or 1 mM furosemide (Na+/K+/2Cl− inhibitor) were then studied. We found that each of these inhibitors nearly abolished H2O2 induced increases in intracellular Na+. Western blot analysis of isolated mTAL from SD rats for each of the treatments found that 100 uM H2O2 increased the activation of AKT (pAKTSer473; a surrogate for mTORC1/2 activation) and S6 (pS6Ser235/236; a surrogate for mTORC1 activation) and were inhibited by their respective inhibitors.ConclusionTogether these observations indicate that H2O2 acts through the mTOR signaling pathways to inhibit NKCC2 activity, which would in turn be expected to lead to sodium retention and hypertension.Support or Funding InformationHL 122662This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
α‐actinin 4 Knockdown Reduced Vasopressin‐Induced Aquaporin‐2 Expression in the Kidney Collecting Duct Cells
Authors: Ho Cheng Hsuan , Yu Ming‐Jiun 
Published: June 2021

Vasopressin is a pituitary peptide hormone that regulates aquaporin‐2 (AQP2) gene expression in the kidney collecting duct principal cells. Dysregulation of vasopressin‐regulated AQP2 gene expression is associated with many water balance disorders. Previously in the collecting duct cell model (mpkCCD), we identified α actinin 4, a cytoskeletal actin‐binding protein that was reported to translocate into the nucleus and acts as a transcriptional co‐activator. Here, we found that short hairpin RNA (shRNA)‐mediated α actinin 4 knockdown in the mpkCCD cells reduced vasopressin‐induced AQP2 mRNA and protein levels, suggesting a role of α actinin 4 in vasopressin‐induced AQP2 gene expression. However, vasopressin did not induce nuclear translocation of α actinin 4, suggesting a different model of α actinin 4 action. α actinin 4 was reported as a transcriptional co‐activator of the glucocorticoid receptor whose agonist dexamethasone was shown to enhance vasopressin‐induced AQP2 gene expression in the mpkCCD cells in a dose‐ and time‐dependent manner. We thus tested whether α actinin 4 interacts with glucocorticoid receptor to enhance vasopressin‐induced AQP2 gene expression. In support of our hypothesis, shRNA‐mediated glucocorticoid receptor knockdown almost blunted vasopressin‐induced AQP2 mRNA and protein expression. Our results are consistent with a role of α actinin 4 in vasopressin‐induced AQP2 gene expression involving glucocorticoid receptor.Support or Funding InformationThis work was supported by Ministry of Science and Technology, Taiwan to MJY (MOST104‐2320‐B002‐064‐MY3)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effectiveness of Total Water Intake Guidelines in Maintaining Lowered Urine Osmolality
Authors: Seal Adam , Johnson Evan , Pérronet Francois , Jansen Lisa , Summers LynnDee , Adams J.D. , Perrier Erica , Guelinckx Isabelle , Kavouras Stavros 
Published: June 2021

The reference values for daily total water intake from fluids and foods (TWI) from the Institute of Medicine (IOM) are 2.7 and 3.7 liters for females and males, respectively. Recent studies suggest that for optimal water intake 24h urine osmolality should be maintained below 500 mmol·kg−1.PurposeTo determine the total water intake requirement for healthy adults to maintain 24h urine osmolality below 500 mmol·kg−1.MethodsUrinary concentration was assessed in 48 men and 50 women (age: 41±14 y, body mass index: 26.2±5.1 kg·m−2) via urine osmolality from 24h samples (UOsm). TWI was assessed via water turnover using the deuterium dilution (D2O) method throughout a 7‐day time period, corrected for metabolic water production. The diagnostic accuracy of TWI to identify UOsm ≤ 500 mmol·kg−1 was evaluated using receiver operating characteristic (ROC) analysis in men and women separately.ResultsUOsm was 511±232 and 402±203 mmol·kg−1 and TWI was 3.54±1.08 L·d−1 and 3.20±1.27 L·d−1 in men and women, respectively. 32% of men and 55% of women met IOM guidelines for TWI. ROC analysis for TWI detecting UOsm ≤ 500 mmol·kg−1 in men yielded an area under the curve (AUC) of 77.4% with sensitivity, specificity, and threshold values of 83.3%, 64.5%, and 3.39 L·d−1, respectively. In women, the AUC was 82.4% with sensitivity, specificity, and threshold values of 85.7%, 72.1%, and 2.61 L·d−1, respectively.ConclusionConsidering threshold values in men and women of 3.39 L·d−1 and 2.61 L·d−1, respectively, maintaining TWI in line with IOM guidelines of 3.7 L·d−1 in men and 2.7 L·d−1 in women should be sufficient for most individuals to maintain 24h urine osmolality less than 500 mmol·kg−1.Support or Funding InformationThis research was supported by Danone Research.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.