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At least 1,585 records · Page 88
All sourcesNASA NTRSDOE OSTI
DOE OSTI2013

Toxicodynamics of rigid polystyrene microparticles on pulmonary gas exchange in mice: Implications for microemboli-based drug delivery systems

The toxicodynamic relationship between the number and size of pulmonary microemboli resulting from uniformly sized, rigid polystyrene microparticles (MPs) administered intravenously and their potential effects on pulmonary gas exchange were investigated. CD-1 male mice (6–8 weeks) were intravenously administered 10, 25 and 45 μm diameter MPs. Oxygen hemoglobin saturation in the blood (SpO{sub 2}) was measured non-invasively using a pulse oximeter while varying inhaled oxygen concentration (F{sub I}O{sub 2}). The resulting data were fit to a physiologically based non-linear mathematical model that estimates 2 parameters: ventilation–perfusion ratio (V{sub A}/Q) and shunt (percentage of deoxygenated blood returning to systemic circulation). The number of MPs administered prior to a statistically significant reduction in normalized V{sub A}/Q was dependent on particle size. MP doses that resulted in a significant reduction in normalized V{sub A}/Q one day post-treatment were 4000, 40,000 and 550,000 MPs/g for 45, 25 and 10 μm MPs, respectively. The model estimated V{sub A}/Q and shunt returned to baseline levels 7 days post-treatment. Measuring SpO{sub 2} alone was not sufficient to observe changes in gas exchange; however, when combined with model-derived V{sub A}/Q and shunt early reversible toxicity from pulmonary microemboli was detected suggesting that the model and physical measurements are both required for assessing toxicity. Moreover, it appears that the MP load required to alter gas exchange in a mouse prior to lethality is significantly higher than the anticipated required MP dose for effective drug delivery. Overall, the current results indicate that the microemboli-based approach for targeted pulmonary drug delivery is potentially safe and should be further explored. -- Highlights: ► Murine pulmonary gas exchange after microembolization was non-invasively studied. ► A physiologically based model quantified impairment of pulmonary gas exchange. ► Number and size of microemboli determine severity of impaired gas exchange. ► Pulmonary gas exchange returns to baseline within 7 days.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Safflor yellow B suppresses angiotensin II-mediated human umbilical vein cell injury via regulation of Bcl-2/p22{sup phox} expression

Intracellular reactive oxygen species (ROS) are derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Angiotensin II (Ang II) can cause endothelial dysfunction by promoting intracellular ROS generation. Safflor yellow B (SYB) effectively inhibits ROS generation by upregulating Bcl-2 expression. In this study, we examined the effects of SYB on Ang II-induced injury to human umbilical vein endothelial cells (HUVECs), and elucidated the roles of NADPH oxidase and Bcl-2. We treated cultured HUVECs with Ang II, SYB, and Bcl-2 siRNA, and determined NADPH oxidase activity and ROS levels. Furthermore, cellular and mitochondrial physiological states were evaluated, and the expression levels of target proteins were analyzed. Ang II significantly enhanced intracellular ROS levels, caused mitochondrial membrane dysfunction, and decreased cell viability, leading to apoptosis. This was associated with increased expression of AT1R and p22{sup phox}, increased NADPH oxidase activity, and an increased ratio of Bax/Bcl-2, leading to decreases in antioxidant enzyme activities, which were further strengthened after blocking Bcl-2. Compared to Ang II treatment alone, co-treatment with SYB significantly reversed HUVEC injury. Taken together, these results demonstrate that SYB could significantly protect endothelial cells from Ang II-induced cell damage, and that it does so by upregulating Bcl-2 expression and inhibiting ROS generation. - Highlights: • Angiotensin II depresses mitochondria physiological function. • Angiotensin II activates NADPH oxidase via up-regulating expresion of p22{sup phox}. • Bcl-2 plays a pivotal role in improving mitochondria function and regulates ROS level. • Inhibitor of Bcl-2 promotes angiotensin II mediated HUVEC injury. • SYB attenuates angiotensin II mediated HUVEC injury via up regulating Bcl-2 expression.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Assessment of beating parameters in human induced pluripotent stem cells enables quantitative in vitro screening for cardiotoxicity

Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes show promise for screening during early drug development. Here, we tested a hypothesis that in vitro assessment of multiple cardiomyocyte physiological parameters enables predictive and mechanistically-interpretable evaluation of cardiotoxicity in a high-throughput format. Human iPSC-derived cardiomyocytes were exposed for 30 min or 24 h to 131 drugs, positive (107) and negative (24) for in vivo cardiotoxicity, in up to 6 concentrations (3 nM to 30 uM) in 384-well plates. Fast kinetic imaging was used to monitor changes in cardiomyocyte function using intracellular Ca{sup 2+} flux readouts synchronous with beating, and cell viability. A number of physiological parameters of cardiomyocyte beating, such as beat rate, peak shape (amplitude, width, raise, decay, etc.) and regularity were collected using automated data analysis. Concentration–response profiles were evaluated using logistic modeling to derive a benchmark concentration (BMC) point-of-departure value, based on one standard deviation departure from the estimated baseline in vehicle (0.3% dimethyl sulfoxide)-treated cells. BMC values were used for cardiotoxicity classification and ranking of compounds. Beat rate and several peak shape parameters were found to be good predictors, while cell viability had poor classification accuracy. In addition, we applied the Toxicological Prioritization Index (ToxPi) approach to integrate and display data across many collected parameters, to derive “cardiosafety” ranking of tested compounds. Multi-parameter screening of beating profiles allows for cardiotoxicity risk assessment and identification of specific patterns defining mechanism-specific effects. These data and analysis methods may be used widely for compound screening and early safety evaluation in drug development. - Highlights: • Induced pluripotent stem cell-derived cardiomyocytes are promising in vitro models. • We tested if evaluation of cardiotoxicity is possible in a high-throughput format. • The assay shows benefits of automated data integration across multiple parameters. • Quantitative assessment of concentration–response is possible using iPSCs. • Multi-parametric screening allows for cardiotoxicity risk assessment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Adverse testicular effects of Botox® in mature rats

Botox® injections are taking a consistently increasing place in urology. Intracremasteric injections, particularly, have been applied for cryptorchidism and painful testicular spasms. Studies outlining their safety for this use are, however, scanty. Thus, the present study aimed at evaluating possible testicular toxicity of Botox® injections and their effect on male fertility. Mature rats were given intracremasteric Botox® injections (10, 20 and 40 U/kg) three times in a two-week interval. Changes in body and testes weights were examined and gonadosomatic index compared to control group. Semen quality, sperm parameters, fructose, protein, cholesterol and triglycerides contents were assessed. Effects on normal testicular function were investigated by measuring testosterone levels and changes in enzyme activities (lactate dehydrogenase-X and acid phosphatase). To draw a complete picture, changes in oxidative and inflammatory states were examined, in addition to the extent of connective tissue deposition between seminiferous tubules. In an attempt to have more accurate information about possible spermatotoxic effects of Botox®, flowcytometric analysis and histopathological examination were carried out. Botox®-injected rats showed altered testicular physiology and function. Seminiferous tubules were separated by dense fibers, especially with the highest dose. Flowcytometric analysis showed a decrease in mature sperms and histopathology confirmed the findings. The oxidative state was, however, comparable to control group. This study is the first to show that intracremasteric injections of Botox® induce adverse testicular effects evidenced by inhibited spermatogenesis and initiation of histopathological changes. In conclusion, decreased fertility may be a serious problem Botox® injections could cause. - Highlights: • Botox® injections are the trend nowadays, for both medical and non-medical uses. • They were recently suggested for cryptorchidism and testicular spasms. • This study outlines possible testicular adverse effects of these injections. • Botox® affected normal testicular function and physiology. • Infertility is a serious problem that Botox® injections could cause.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Effect of the combination of metformin hydrochloride and melatonin on oxidative stress before and during pregnancy, and biochemical and histopathological analysis of the livers of rats after treatment for polycystic ovary syndrome

The aim of the present study was to analyze the effect of a combination of metformin hydrochloride and melatonin on oxidative stress together with a biochemical and histopathological analysis of the livers of Wistar rats induced with PCOS. The results indicated that a combination of the drugs was more effective in the reduction of plasmatic levels of liver enzyme alanine aminotransferase, nitric oxide and total glutathione, and decreased the inflammatory response and histopathological damage, producing results that were significantly similar to animals from the control group. A mixture of the drugs produced more effective results against liver toxicity caused by PCOS, encouraging the normalization of biochemical parameters. During pregnancy, there was reduced oxidative stress compared to monotherapeutic use of these drugs. Interestingly, the combination of the drugs caused a physiological reaction similar to responses identified in healthy rats without induction of the PCOS control group. However, the clinical and physiological effectiveness of the combination should be further explored, especially with respect to the possible side effects on offspring. - Highlights: • Studies have documented increased oxidative stress in patients with PCOS. • It has been noted that women with PCOS have a high prevalence of liver alterations. • Liver disease in pregnancy may be pre-existing increasing the newborn mortality. • Metformin/melatonin associated reduced oxidative stress in liver in pregnant rats. • Association of metformin/melatonin normalizes hepatic biochemical parameters.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Selectivity of natural, synthetic and environmental estrogens for zebrafish estrogen receptors

Zebrafish, Danio rerio, is increasingly used as an animal model to study the effects of pharmaceuticals and environmental estrogens. As most of these estrogens have only been tested on human estrogen receptors (ERs), it is necessary to measure their effects on zebrafish ERs. In humans there are two distinct nuclear ERs (hERα and hERβ), whereas the zebrafish genome encodes three ERs, zfERα and two zfERβs (zfERβ1 and zfERβ2). In this study, we established HeLa-based reporter cell lines stably expressing each of the three zfERs. We first reported that estrogens more efficiently activate the zfERs at 28 °C as compared to 37 °C, thus reflecting the physiological temperature of zebrafish in wildlife. We then showed significant differences in the ability of agonist and antagonist estrogens to modulate activation of the three zfER isotypes in comparison to hERs. Environmental compounds (bisphenol A, alkylphenols, mycoestrogens) which are hER panagonists and hERβ selective agonists displayed greater potency for zfERα as compared to zfERβs. Among hERα selective synthetic agonists, PPT did not activate zfERα while 16α-LE2 was the most zfERα selective compound. Altogether, these results confirm that all hER ligands control in a similar manner the transcriptional activity of zfERs although significant differences in selectivity were observed among subtypes. The zfER subtype selective ligands that we identified thus represent new valuable tools to dissect the physiological roles of the different zfERs. Finally, our work also points out that care has to be taken in transposing the results obtained using the zebrafish as a model for human physiopathology. - Highlights: • Zebrafish is increasingly used to study the effects of estrogens. • We assessed the activity of pharmaceutical and environmental estrogens on zfERs. • Environmental estrogens displayed greater potency for zfERα compared to zfERβs. • hERβ selective agonists displayed greater potency for zfERα compared to zfERβs. • The hERα selective agonist 16αL-E2 is the most zfERα selective compound.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Fibroblasts maintained in 3 dimensions show a better differentiation state and higher sensitivity to estrogens

Cell differentiation and response to hormonal signals were studied in a 3D environment on an in-house generated mouse fibroblast cell line expressing a reporter gene under the control of estrogen responsive sequences (EREs). 3D cell culture conditions were obtained in a Rotary Cell Culture System; (RCCS™), a microgravity based bioreactor that promotes the aggregation of cells into multicellular spheroids (MCS). In this bioreactor the cells maintained a better differentiated phenotype and more closely resembled in vivo tissue. The RCCS™ cultured fibroblasts showed higher expression of genes regulating cell assembly, differentiation and hormonal functions. Microarray analysis showed that genes related to cell cycle, proliferation, cytoskeleton, migration, adhesion and motility were all down-regulated in 3D as compared to 2D conditions, as well as oncogene expression and inflammatory cytokines. Controlled remodeling of ECM, which is an essential aspect of cell organization, homeostasis and tissue was affected by the culture method as assessed by immunolocalization of β-tubulin. Markers of cell organization, homeostasis and tissue repair, metalloproteinase 2 (MMP2) and its physiological inhibitor (TIMP4) changed expression in association with the relative formation of cell aggregates. The fibroblasts cultured in the RCCS™ maintain a better responsiveness to estrogens, measured as expression of ERα and regulation of an ERE-dependent reporter and of the endogenous target genes CBP, Rarb, MMP1 and Dbp. Our data highlight the interest of this 3D culture model for its potential application in the field of cell response to hormonal signals and the pharmaco-toxicological analyses of chemicals and natural molecules endowed of estrogenic potential. - Highlights: • We here characterized the first cell line derived from an estrogen reporter mouse. • In the RCCS cells express an immortalized behavior but not a transformed phenotype. • The RCCS provides a system for maintaining cells in more physiological conditions. • RCCS-cultured fibroblasts showed higher hormonal sensitivity to estradiol. • This bioreactor is a novel 3D model to be applied to pharmacotoxicological studies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

An integrated QSAR-PBK/D modelling approach for predicting detoxification and DNA adduct formation of 18 acyclic food-borne α,β-unsaturated aldehydes

Acyclic α,β-unsaturated aldehydes present in food raise a concern because the α,β-unsaturated aldehyde moiety is considered a structural alert for genotoxicity. However, controversy remains on whether in vivo at realistic dietary exposure DNA adduct formation is significant. The aim of the present study was to develop physiologically based kinetic/dynamic (PBK/D) models to examine dose-dependent detoxification and DNA adduct formation of a group of 18 food-borne acyclic α,β-unsaturated aldehydes without 2- or 3-alkylation, and with no more than one conjugated double bond. Parameters for the PBK/D models were obtained using quantitative structure–activity relationships (QSARs) defined with a training set of six selected aldehydes. Using the QSARs, PBK/D models for the other 12 aldehydes were defined. Results revealed that DNA adduct formation in the liver increases with decreasing bulkiness of the molecule especially due to less efficient detoxification. 2-Propenal (acrolein) was identified to induce the highest DNA adduct levels. At realistic dietary intake, the predicted DNA adduct levels for all aldehydes were two orders of magnitude lower than endogenous background levels observed in disease free human liver, suggesting that for all 18 aldehydes DNA adduct formation is negligible at the relevant levels of dietary intake. The present study provides a proof of principle for the use of QSAR-based PBK/D modelling to facilitate group evaluations and read-across in risk assessment. - Highlights: • Physiologically based in silico models were made for 18 α,β-unsaturated aldehydes. • Kinetic parameters were determined by in vitro incubations and a QSAR approach. • DNA adduct formation was negligible at levels relevant for dietary intake. • The use of QSAR-based PBK/D modelling facilitates group evaluations and read-across.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

In vitro metabolism of benzo[a]pyrene-7,8-dihydrodiol and dibenzo[def,p]chrysene-11,12 diol in rodent and human hepatic microsomes

Polycyclic aromatic hydrocarbons (PAHs) are contaminants that are ubiquitously found in the environment, produced through combustion of organic matter or petrochemicals, and many of which are procarcinogens. The prototypic PAH, benzo[a]pyrene (B[a]P) and the highly carcinogenic dibenzo[def,p]chrysene (DBC) are metabolically activated by isoforms of the P450 enzyme superfamily producing benzo[a]pyrene-7,8-dihydrodiol (B[a]P diol), dibenzo[def,p]chrysene-11,12 diol (DBC diol). Each of these diols can be further metabolized by cytochrome P450 enzymes to highly reactive diol-epoxide metabolites that readily react with DNA or by phase II conjugation facilitating excretion. To complement prior in vitro metabolism studies with parent B[a]P and DBC, both phase I metabolism and phase II glucuronidation of B[a]P diol and DBC diol were measured in hepatic microsomes from female B6129SF1/J mice, male Sprague-Dawley rats, and female humans. Metabolic parameters, including intrinsic clearance and Michaelis-Menten kinetics were calculated from substrate depletion data. Mice and rats demonstrated similar B[a]P diol phase I metabolic rates. Compared to rodents, human phase I metabolism of B[a]P diol demonstrated lower overall metabolic capacity, lower intrinsic clearance at higher substrate concentrations (>0.14 µM), and higher intrinsic clearance at lower substrate concentrations (<0.07 µM). Rates of DBC diol metabolism did not saturate in mice or humans and were highest overall in mice. Higher affinity constants and lower capacities were observed for DBC diol glucuronidation compared to B[a]P diol glucuronidation; however, intrinsic clearance values for these compounds were consistent within each species. Kinetic parameters reported here will be used to extend physiologically based pharmacokinetic (PBPK) models to include the disposition of B[a]P and DBC metabolites in animal models and humans to support future human health risk assessments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Feed Your Friends: Do Plant Exudates Shape the Root Microbiome?

Plant health in natural environments depends on interactions with complex and dynamic communities comprising macro- and microorganisms. While many studies have provided insights into the composition of rhizosphere microbiomes (rhizobiomes), little is known about whether plants shape their rhizobiomes. In this work, we discuss physiological factors of plants that may govern plant–microbe interactions, focusing on root physiology and the role of root exudates. Given that only a few plant transport proteins are known to be involved in root metabolite export, we suggest novel families putatively involved in this process. Finally, building off of the features discussed in this review, and in analogy to well-known symbioses, we elaborate on a possible sequence of events governing rhizobiome assembly. Recent advances in sequencing technology have been enabling high-throughput characterization of highly complex plant-associated microbial communities, which are relevant for plant health. Metagenomic approaches have been identifying the metabolic potential of microbes, and are starting to reveal functional groups of microbes, reducing the overall complexity of rhizobiomes. Metabolomic studies are uncovering differential root exudation in various environments and plant developmental stages, as well as consumption of specific exometabolites by microbes. Genome-wide association studies and other comparative genomic approaches have been revealing a link between plant genetic factors, exudation profiles, and microbiome compositions. Finally, molecular studies have been characterizing plant transport proteins necessary for interactions with specific microbes, although the transport of most nutrients and signaling molecules remains unexplored.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2011

The origin of pre-neoplastic metaplasia in the stomach: Chief cells emerge from the Mist

The digestive-enzyme secreting, gastric epithelial chief (zymogenic) cell is remarkable and underappreciated. Here, we discuss how all available evidence suggests that mature chief cells in the adult, mammalian stomach are postmitotic, slowly turning over cells that arise via a relatively long-lived progenitor, the mucous neck cell, The differentiation of chief cells from neck cells does not involve cell division, and the neck cell has its own distinct pattern of gene expression and putative physiological function. Thus, the ontogeny of the normal chief cell lineage exemplifies transdifferentiation. Furthermore, under pathophysiogical loss of acid-secreting parietal cell, the chief cell lineage can itself trasndifferentiate into a mucous cell metaplasia designated Spasmolytic Polypeptide Expressing Metaplasia (SPEM). Especially in the presence of inflammation, this metaplastic lineage can regain proliferative capacity and, in humans may also further differentiate into intestinal metaplasia. The results indicate that gastric fundic lineages display remarkable plasticity in both physiological ontogeny and pathophysiological pre-neoplastic metaplasia.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

ALS/FTLD-linked TDP-43 regulates neurite morphology and cell survival in differentiated neurons

Tar-DNA binding protein of 43 kDa (TDP-43) has been characterized as a major component of protein aggregates in brains with neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). However, physiological roles of TDP-43 and early cellular pathogenic effects caused by disease associated mutations in differentiated neurons are still largely unknown. Here, we investigated the physiological roles of TDP-43 and the effects of missense mutations associated with diseases in differentiated cortical neurons. The reduction of TDP-43 by siRNA increased abnormal neurites and decreased cell viability. ALS/FTLD-associated missense mutant proteins (A315T, Q331K, and M337V) were partially mislocalized to the cytosol and neurites when compared to wild-type and showed abnormal neurites similar to those observed in cases of loss of TDP-43. Interestingly, cytosolic expression of wild-type TDP-43 with mutated nuclear localization signals also induced abnormal neurtie morphology and reduction of cell viability. However, there was no significant difference in the effects of cytosolic expression in neuronal morphology and cell toxicity between wild-type and missense mutant proteins. Thus, our results suggest that mislocalization of missense mutant TDP-43 may contribute to loss of TDP-43 function and affect neuronal morphology, probably via dominant negative action before severe neurodegeneration in differentiated cortical neurons. Highlights: • The function of nuclear TDP-43 in neurite morphology in mature neurons. • Partial mislocalization of TDP-43 missense mutants into cytosol from nucleus. • Abnormal neurite morphology caused by missense mutants of TDP-43. • The effect of cytosolic expression of TDP-43 in neurite morphology and in cell survival.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Molecular mechanisms for inhibition of colon cancer cells by combined epigenetic-modulating epigallocatechin gallate and sodium butyrate

Bioactive compounds are considered safe and have been shown to alter genetic and epigenetic profiles of tumor cells. However, many of these changes have been reported at molecular concentrations higher than physiologically achievable levels. We investigated the role of the combinatorial effects of epigallocatechin gallate (EGCG), a predominant polyphenol in green tea, and sodium butyrate (NaB), a dietary microbial fermentation product of fiber, in the regulation of survivin, which is an overexpressed anti-apoptotic protein in colon cancer cells. For the first time, our study showed that the combination treatment induced apoptosis and cell cycle arrest in RKO, HCT-116 and HT-29 colorectal cancer cells. This was found to be regulated by the decrease in HDAC1, DNMT1, survivin and HDAC activity in all three cell lines. A G2/M arrest was observed for RKO and HCT-116 cells, and G1 arrest for HT-29 colorectal cancer cells for combinatorial treatment. Further experimentation of the molecular mechanisms in RKO colorectal cancer (CRC) cells revealed a p53-dependent induction of p21 and an increase in nuclear factor kappa B (NF-κB)-p65. An increase in double strand breaks as determined by gamma-H2A histone family member X (γ-H2AX) protein levels and induction of histone H3 hyperacetylation was also observed with the combination treatment. Further, we observed a decrease in global CpG methylation. Taken together, these findings suggest that at low and physiologically achievable concentrations, combinatorial EGCG and NaB are effective in promoting apoptosis, inducing cell cycle arrest and DNA-damage in CRC cells. - Highlights: • EGCG and NaB as a combination inhibits colorectal cancer cell proliferation. • The combination treatment induces DNA damage, G2/M and G1 arrest and apoptosis. • Survivin is effectively down-regulated by the combination treatment. • p21 and p53 expressions are induced by the combination treatment. • Epigenetic proteins DNMT1 and HDAC1 are effectively down-regulated by the treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Stabilization of gene expression and cell morphology after explant recycling during fin explant culture in goldfish

The development of fin primary cell cultures for in vitro cellular and physiological studies is hampered by slow cell outgrowth, low proliferation rate, poor viability, and sparse cell characterization. Here, we investigated whether the recycling of fresh explants after a first conventional culture could improve physiological stability and sustainability of the culture. The recycled explants were able to give a supplementary cell culture showing faster outgrowth, cleaner cell layers and higher net cell production. The cells exhibited a highly stabilized profile for marker gene expression including a low cytokeratin 49 (epithelial marker) and a high collagen 1a1 (mesenchymal marker) expression. Added to the cell spindle-shaped morphology, motility behavior, and actin organization, this suggests that the cells bore stable mesenchymal characteristics. This contrast with the time-evolving expression pattern observed in the control fresh explants during the first 2 weeks of culture: a sharp decrease in cytokeratin 49 expression was concomitant with a gradual increase in col1a1. We surmise that such loss of epithelial features for the benefit of mesenchymal ones was triggered by an epithelial to mesenchymal transition (EMT) process or by way of a progressive population replacement process. Overall, our findings provide a comprehensive characterization of this new primary culture model bearing mesenchymal features and whose stability over culture time makes those cells good candidates for cell reprogramming prior to nuclear transfer, in a context of fish genome preservation. - Highlights: • Recycled fin explants outgrow cells bearing stable mesenchymal traits. • Cell production and quality is enhanced in the recycled explant culture system. • Fresh fin primary culture is highly variable and loose epithelial traits over time.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

On the evolution of the pulmonary alveolar lipofibroblast

The pulmonary alveolar lipofibroblast was first reported in 1970. Since then its development, structure, function and molecular characteristics have been determined. Its capacity to actively absorb, store and ‘traffic’ neutral lipid for protection of the alveolus against oxidant injury, and for the active supply of substrate for lung surfactant phospholipid production have offered the opportunity to identify a number of specialized functions of these strategically placed cells. Namely, Parathyroid Hormone-related Protein (PTHrP) signaling, expression of Adipocyte Differentiation Related Protein, leptin, peroxisome proliferator activator receptor gamma, and the prostaglandin E2 receptor EP2- which are all stretch-regulated, explaining how and why surfactant production is ‘on-demand’ in service to ventilation–perfusion matching. Because of the central role of the lipofibroblast in vertebrate lung physiologic evolution, it is a Rosetta Stone for understanding how and why the lung evolved in adaptation to terrestrial life, beginning with the duplication of the PTHrP Receptor some 300 mya. Moreover, such detailed knowledge of the workings of the lipofibroblast have provided insight to the etiology and effective treatment of Bronchopulmonary Dysplasia based on physiologic principles rather than on pharmacology. - Highlights: • development and function of the pulmonary alveolar lipofibroblast phenotype. • Regulation of lipofibroblast neutral lipid metabolism. • Exploiting the deep homologies regulating lipofibroblast structure and function. • Utilizing such in-depth knowledge to understand the etiology and effective treatment of Bronchopulmonary Dysplasia.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Modulation of the uptake of critical nutrients by breast cancer cells by lactate: Impact on cell survival, proliferation and migration

This work aimed to characterize the uptake of folate and glucose by breast cancer cells and to study the effect of lactate upon the transport of these nutrients and upon cell viability, proliferation and migration capacity. Data obtained showed that: a) MCF7 cells uptake {sup 3}H-folic acid ({sup 3}H-FA) at physiological but not at acidic pH; b) T47D cells accumulate {sup 3}H-FA and {sup 14}C-5-methyltetrahydrofolate ({sup 14}C-5-MTHF) more efficiently at acidic than at physiological pH; c) {sup 3}H-deoxyglucose ({sup 3}H-DG) uptake by T47D cells is sodium-independent, inhibited by cytochalasin B (CYT B) and stimulated by insulin. Regarding the effect of lactate, in T47D cells, acute (26 min) and chronic (24 h) exposure to lactic acid (LA) stimulated {sup 3}H-FA uptake. Acute exposure to LA also stimulated {sup 3}H-DG uptake and chronic exposure to LA significantly stimulated T47D cell migratory capacity. In conclusion, the transport of folates is strikingly different in two phenotypically similar breast cancer cell lines: MCF7 and T47D cells. Additionally, lactate seems to act as a signaling molecule which increases the uptake of nutrients and promotes the migration capacity of T47D cells. - Highlights: • Transport of folates is strikingly different in MCF7 and T47D cells. • Lactate increases the uptake of folate and glucose by T47D cells. • Lactate promotes T47D cell migratory capacity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Vitamin A and insulin are required for the maintenance of hepatic stellate cell quiescence

Transdifferentiation of vitamin A-storing hepatic stellate cells (HSCs) to vitamin A-depleted myofibroblastic cells leads to liver fibrosis. Vitamin A regulates lipid accumulation and gene transcription, suggesting that vitamin A is involved in the maintenance of HSC quiescence under a physiological condition. However, the precise mechanism remains elusive because there is no appropriate in vitro culture system for quiescent HSCs. Here, we show that treatment of quiescent HSCs with vitamin A partially maintained the accumulation of lipid droplets and expression of quiescent HSC markers (glial fibrillary acidic protein, peroxisome proliferator-activator receptor-γ and CCAAT/enhancer-binding protein-α) and also the expression of myofibroblastic markers (α-smooth muscle actin, heat shock protein 47 and collagen type I). On the other hand, combined treatment with vitamin A and insulin sustained the characteristic of HSC quiescence and completely suppressed the expression of myofibroblastic markers through activation of the JAK2/STAT5 signaling pathway and increased expression of sterol regulatory element binding protein-1. These treated HSCs transdifferentiated to myofibroblastic cells under a culture condition with fetal bovine serum. The results suggest an important role of vitamin A and insulin in the maintenance of HSC quiescence under a physiological condition. - Highlights: • Vitamin A partially maintained the quiescent phenotypes of HSCs. • Combined treatment with vitamin A and insulin sustained HSC quiescence. • These treated HSCs activated under a culture condition with FBS. • HSC quiescence is maintained by activation of JAK2/STAT5 and SREBP-1 expression.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Functional transferred DNA within extracellular vesicles

Extracellular vesicles (EVs) are small membrane vesicles including exosomes and shedding vesicles that mediated a cell-to-cell communication. EVs are released from almost all cell types under both physiological and pathological conditions and incorporate nuclear and cytoplasmic molecules for intercellular delivery. Besides protein, mRNA, and microRNA of these molecules, as recent studies show, specific DNA are prominently packaged into EVs. It appears likely that some of exosomes or shedding vesicles, bearing nuclear molecules are released upon bubble-like blebs. Specific interaction of EVs with susceptible recipients performs the uptake of EVs into the target cells, discharging their cargo including nuclear and cytoplasmic macromolecules into the cytosol. These findings expand the nucleic acid content of EVs to include increased levels of specific DNA. Thus, EVs contain a repertoire of genetic information available for horizontal gene transfer and potential use as blood biomarkers for cancer and atherosclerosis. In this review, the focus is on the characteristics, biological functions, and roles in diseases of DNA within EVs. - Highlights: • This review is focused on the DNA within EVs including its characteristics, biological functions, and roles in diseases. • It is clear that DNA within EVs might have important physiological and pathological roles in various diseases. • Knowledge in this area may provides us alternative methods for disease diagnosis or therapy in the future.

60 APPLIED LIFE SCIENCES↗