Coregulation of FANCA and BRCA1 in human cells.

PubMed

Haitjema, Anneke; Mol, Berber M; Kooi, Irsan E; Massink, Maarten Pg; Jørgensen, Jens Al; Rockx, Davy Ap; Rooimans, Martin A; de Winter, Johan P; Meijers-Heijboer, Hanne; Joenje, Hans; Dorsman, Josephine C

2014-01-01

Fanconi anemia (FA) is a genetically heterogeneous syndrome associated with increased cancer predisposition. The underlying genes govern the FA pathway which functions to protect the genome during the S-phase of the cell cycle. While upregulation of FA genes has been linked to chemotherapy resistance, little is known about their regulation in response to proliferative stimuli. The purpose of this study was to examine how FA genes are regulated, especially in relation to the cell cycle, in order to reveal their possible participation in biochemical networks. Expression of 14 FA genes was monitored in two human cell-cycle models and in two RB1/E2F pathway-associated primary cancers, retinoblastoma and basal breast cancer. In silico studies were performed to further evaluate coregulation and identify connected networks and diseases. Only FANCA was consistently induced over 2-fold; FANCF failed to exhibit any regulatory fluctuations. Two tools exploiting public data sets indicated coregulation of FANCA with BRCA1. Upregulation of FANCA and BRCA1 correlated with upregulation of E2F3. Genes coregulated with both FANCA and BRCA1 were enriched for MeSH-Term id(s) genomic instability, microcephaly, and Bloom syndrome, and enriched for the cellular component centrosome. The regulation of FA genes appears highly divergent. In RB1-linked tumors, upregulation of FA network genes was associated with reduced expression of FANCF. FANCA and BRCA1 may jointly act in a subnetwork - supporting vital function(s) at the subcellular level (centrosome) as well as at the level of embryonic development (mechanisms controlling head circumference).

Fanconi anemia (FA) binding protein FAAP20 stabilizes FA complementation group A (FANCA) and participates in interstrand cross-link repair.

PubMed

Leung, Justin Wai Chung; Wang, Yucai; Fong, Ka Wing; Huen, Michael Shing Yan; Li, Lei; Chen, Junjie

2012-03-20

The Fanconi anemia (FA) pathway participates in interstrand cross-link (ICL) repair and the maintenance of genomic stability. The FA core complex consists of eight FA proteins and two Fanconi anemia-associated proteins (FAAP24 and FAAP100). The FA core complex has ubiquitin ligase activity responsible for monoubiquitination of the FANCI-FANCD2 (ID) complex, which in turn initiates a cascade of biochemical events that allow processing and removal of cross-linked DNA and thereby promotes cell survival following DNA damage. Here, we report the identification of a unique component of the FA core complex, namely, FAAP20, which contains a RAD18-like ubiquitin-binding zinc-finger domain. Our data suggest that FAAP20 promotes the functional integrity of the FA core complex via its direct interaction with the FA gene product, FANCA. Indeed, somatic knockout cells devoid of FAAP20 displayed the hallmarks of FA cells, including hypersensitivity to DNA cross-linking agents, chromosome aberrations, and reduced FANCD2 monoubiquitination. Taking these data together, our study indicates that FAAP20 is an important player involved in the FA pathway.

Antisense Down-Regulation of the FaPG1 Gene Reveals an Unexpected Central Role for Polygalacturonase in Strawberry Fruit Softening1[W

PubMed Central

Quesada, Miguel A.; Blanco-Portales, Rosario; Posé, Sara; García-Gago, Juan A.; Jiménez-Bermúdez, Silvia; Muñoz-Serrano, Andrés; Caballero, José L.; Pliego-Alfaro, Fernando; Mercado, José A.; Muñoz-Blanco, Juan

2009-01-01

The strawberry (Fragaria × ananassa ‘Chandler’) fruit undergoes a fast softening during ripening. Polygalacturonase (PG) activity is low during this process, but two ripening-related PG genes, FaPG1 and FaPG2, have been cloned. Both genes were up-regulated during fruit ripening and were also negatively regulated by auxin. To further assess the role of FaPG1 on strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the 35S promoter (APG lines) were obtained. Sixteen out of 30 independent transgenic lines showed fruit yields similar to those of the control. Several quality parameters were measured in ripe fruits from these 16 lines. Fruit weight was slightly reduced in four lines, and most of them showed an increase in soluble solid content. Half of these lines yielded fruits significantly firmer than did the control. Four APG lines were selected, their ripened fruits being on average 163% firmer than the control. The postharvest softening of APG fruits was also diminished. Ripened fruits from the four selected lines showed a 90% to 95% decrease in FaPG1 transcript abundance, whereas the level of FaPG2 was not significantly altered. Total PG activity was reduced in three of these lines when compared with control fruits. Cell wall extracts from APG fruits showed a reduction in pectin solubilization and an increase in pectins covalently bound to the cell wall. A comparative transcriptomic analysis of gene expression between the ripened receptacle of the control and those of the APG fruits (comprising 1,250 receptacle expressed sequence tags) did not show any statistically significant change. These results indicate that FaPG1 plays a central role in strawberry softening. PMID:19395408

Fanconi anemia (FA) binding protein FAAP20 stabilizes FA complementation group A (FANCA) and participates in interstrand cross-link repair

PubMed Central

Leung, Justin Wai Chung; Wang, Yucai; Fong, Ka Wing; Huen, Michael Shing Yan; Li, Lei; Chen, Junjie

2012-01-01

The Fanconi anemia (FA) pathway participates in interstrand cross-link (ICL) repair and the maintenance of genomic stability. The FA core complex consists of eight FA proteins and two Fanconi anemia-associated proteins (FAAP24 and FAAP100). The FA core complex has ubiquitin ligase activity responsible for monoubiquitination of the FANCI-FANCD2 (ID) complex, which in turn initiates a cascade of biochemical events that allow processing and removal of cross-linked DNA and thereby promotes cell survival following DNA damage. Here, we report the identification of a unique component of the FA core complex, namely, FAAP20, which contains a RAD18-like ubiquitin-binding zinc-finger domain. Our data suggest that FAAP20 promotes the functional integrity of the FA core complex via its direct interaction with the FA gene product, FANCA. Indeed, somatic knockout cells devoid of FAAP20 displayed the hallmarks of FA cells, including hypersensitivity to DNA cross-linking agents, chromosome aberrations, and reduced FANCD2 monoubiquitination. Taking these data together, our study indicates that FAAP20 is an important player involved in the FA pathway. PMID:22396592

Filter paper-assisted cell transfer (FaCT) technique: A novel cell-sampling technique for intraoperative diagnosis of central nervous system tumors.

PubMed

Kawamura, Jumpei; Kamoshida, Shingo; Shimakata, Takaaki; Hayashi, Yurie; Sakamaki, Kuniko; Denda, Tamami; Kawai, Kenji; Kuwao, Sadahito

2017-04-01

Intraoperative diagnosis of central nervous system (CNS) tumors provides critical guidance to surgeons in the determination of surgical resection margins and treatment. The techniques and preparations used for the intraoperative diagnosis of CNS tumors include frozen sectioning and cytologic methods (squash smear and touch imprint). Cytologic specimens, which do not have freezing artifacts, are important as an adjuvant tool to frozen sections. However, if the amount of submitted tissue samples is limited, then it is difficult to prepare both frozen sections and squash smears or touch imprint specimens from a single sample at the same time. Therefore, the objective of this study was to derive cells directly from filter paper on which tumor samples are placed. The authors established the filter paper-assisted cell transfer (FaCT) smear technique, in which tumor cells are transferred onto a glass slide directly from the filter paper sample spot after the biopsy is removed. Cell yields and diagnostic accuracy of the FaCT smears were assessed in 40 CNS tumors. FaCT smears had ample cell numbers and well preserved cell morphology sufficient for cytologic diagnosis, even if the submitted tissues were minimal. The overall diagnostic concordance rates between frozen sections and FaCT smears were 90% and 87.5%, respectively (no significant differences). When combining FaCT smears with frozen sections, the diagnostic concordance rate rose to 92.5%. The current results suggest that the FaCT smear technique is a simple and effective processing method that has significant value for intraoperative diagnosis of CNS tumors. Cancer Cytopathol 2017;125:277-282. © 2016 American Cancer Society. © 2017 American Cancer Society.

A leu-rich repeat receptor-like protein kinase, FaRIPK1, interacts with the ABA receptor, FaABAR, to regulate fruit ripening in strawberry.

PubMed

Hou, Bing-Zhu; Xu, Cheng; Shen, Yuan-Yue

2018-03-24

Strawberry (Fragaria×ananassa) is a model plant for studying non-climacteric fruit ripening regulated by abscisic acid (ABA); however, its exact molecular mechanisms are yet not fully understood. In this study, a predicted leu-rich repeat (LRR) receptor-like kinase in strawberry, red-initial protein kinase 1 (FaRIPK1), was screened and, using a yeast two-hybrid assay, was shown to interact with a putative ABA receptor, FaABAR. This association was confirmed by bimolecular fluorescence complementation and co-immunoprecipitation assays, and shown to occur in the nucleus. Expression analysis by real-time PCR showed that FaRIPK1 is expressed in roots, stems, leaves, flowers, and fruit, with a particularly high expression in white fruit at the onset of coloration. Down-regulation of FaRIPK1 expression in strawberry fruit, using Tobacco rattle virus-induced gene silencing, inhibited ripening, as evidenced by suppression of ripening-related physiological changes and reduced expression of several genes involved in softening, sugar content, pigmentation, and ABA biosynthesis and signaling. The yeast-expressed LRR and STK (serine/threonine protein kinase) domains of FaRIPK1 bound ABA and showed kinase activity, respectively. A fruit disc-incubation test revealed that FaRIPK1 expression was induced by ABA and ethylene. The synergistic action of FaRIPK1 with FaABAR in regulation of strawberry fruit ripening is discussed.

Correction of both spontaneous and DEB-induced chromosome instability in Fanconi anemia FA-C cells by FACC cDNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stavropoulos, D.J.; Tomkins, D.J.; Allingham-Hawkins, D.J.

1994-09-01

Cells from all four Fanconi anemia complementation groups show hypersensitivity to cell-killing by mitomycin C (MMC), diepoxybutane (DEB) and other DNA cross-linking agents, and increased spontaneous and DEB-induced chromosome aberrations (CA). The extent of these phenotypes varies between lymphoblastoid cell lines from different complementation groups. Our data showed that the difference in MMC hypersensitivity and DEB-CA was not always coupled. While 230N (FA-B) had higher DEB-induced CA/cell than 536N (FA-C) (7.42 vs. 4.46 respectively), that latter was much more sensitive to cell-killing by MMC (dose at 10% survival, D{sub 10}: 5.2 vs. 1.2 ng/ml respectively). Strathdes et al. (1992) clonedmore » a cDNA Fanconi anemia complementation group C (FACC) which complemented the hypersensitivity to MMC and DEB cell-killing of FA-C cells (536N) but not cells from the other three complementation groups. The present study was initiated to determine whether chromosome instability in 536N is also complemented by the FACC (FAC3) cDNA. The pREP4-FAC3 vector was transfected into 536N and transfectants selected with hygromycin B. The DEB D{sub 10} of 536N (1.0 {mu}M) was corrected to the control level (16.2 {mu}M for 3TO) by FACC (15.1 {mu}M for 536N-FACC), as previously demonstrated. Chromosome instability (cab, cse, ctb, cte) was determined without and with 0.1 {mu}g/ml DEB treatment. Spontaneous CA of 536N (0.30 aberrations/cell) was corrected to the control level (0.04 for 3TO) by FACC (0.06 for 536N-FACC). Similarly, the DEB-induced CA was corrected (2.74 for 536N vs. 0.06 and 0.02 for 3TO and 536N-FACC respectively). Thus, at least for FA complementation group C, hypersensitivity to cell-killing and chromosome instability are not dissociated and are most likely caused by the same gene defect.« less

FANCM-FAAP24 and FANCJ: FA proteins that metabolize DNA

PubMed Central

Ali, Abdullah Mahmood; Singh, Thiyam Ramsing; Meetei, Amom Ruhikanta

2009-01-01

Fanconi anemia (FA) is a rare autosomal recessive or X-linked disorder characterized by aplastic anemia, cancer susceptibility and cellular sensitivity to DNA crosslinking agents. Eight FA proteins (FANCA, -B, -C, -E, -F, -G, -L and –M) and three non-FA proteins (FAAP100, FAAP24 and HES1) form the FA nuclear core complex that is required for monoubiquitination of the FANCD2-FANCI dimer upon DNA damage. The other three FA proteins, FANCD1/BRCA2, FANCJ/BACH1/BRIP1 and FANCN/PALB2, act in parallel or downstream of the FANCD2-FANCI dimer. Despite the isolation and characterization of several FA proteins, the mechanism by which these proteins protect cells from DNA interstrand crosslinking agents has been unclear. This is because a majority of the FA proteins lack any recognizable functional domains that can provide insight into their function. The recently discovered FANCM (Hef) and FANCJ (BRIP1/BACH1) proteins contain helicase domains, providing potential insight into the role of FA proteins in DNA repair. FANCM with its partner, FAAP24, and FANCJ bind and metabolize a variety of DNA substrates. In this review, we focus on the discovery, structure, and function of the FANCM-FAAP24 and FANCJ proteins. PMID:19379763

Disruption of the FA/BRCA pathway in bladder cancer.

PubMed

Neveling, K; Kalb, R; Florl, A R; Herterich, S; Friedl, R; Hoehn, H; Hader, C; Hartmann, F H; Nanda, I; Steinlein, C; Schmid, M; Tonnies, H; Hurst, C D; Knowles, M A; Hanenberg, H; Schulz, W A; Schindler, D

2007-01-01

Bladder carcinomas frequently show extensive deletions of chromosomes 9p and/or 9q, potentially including the loci of the Fanconi anemia (FA) genes FANCC and FANCG. FA is a rare recessive disease due to defects in anyone of 13 FANC genes manifesting with genetic instability and increased risk of neoplasia. FA cells are hypersensitive towards DNA crosslinking agents such as mitomycin C and cisplatin that are commonly employed in the chemotherapy of bladder cancers. These observations suggest the possibility of disruption of the FA/BRCA DNA repair pathway in bladder tumors. However, mutations in FANCC or FANCG could not be detected in any of 23 bladder carcinoma cell lines and ten surgical tumor specimens by LOH analysis or by FANCD2 immunoblotting assessing proficiency of the pathway. Only a single cell line, BFTC909, proved defective for FANCD2 monoubiquitination and was highly sensitive towards mitomycin C. This increased sensitivity was restored specifically by transfer of the FANCF gene. Sequencing of FANCF in BFTC909 failed to identify mutations, but methylation of cytosine residues in the FANCF promoter region was demonstrated by methylation-specific PCR, HpaII restriction and bisulfite DNA sequencing. Methylation-specific PCR uncovered only a single instance of FANCF promoter hypermethylation in surgical specimens of further 41 bladder carcinomas. These low proportions suggest that in contrast to other types of tumors silencing of FANCF is a rare event in bladder cancer and that an intact FA/BRCA pathway might be advantageous for tumor progression. Copyright (c) 2007 S. Karger AG, Basel.

Transport and fate of Herbaspirillum chlorophenolicum FA1 in saturated porous media

NASA Astrophysics Data System (ADS)

Li, X.; Xu, H.; Wu, J.

2016-12-01

For the bioremediation of contaminated groundwater, sufficient dispersal of functional microorganisms is one of the most important factors that determine the remediation efficiency. There are extensive studies on the transport of microbes in porous media, while most of them focus on pathogenic bacteria and little attention has been given toward functional bacteria that being used in bioremediation process. Therefore, accurate knowledge of the mechanisms that govern the transport and distribution of such bacteria in groundwater is needed to develop efficient treatment techniques. Herbaspirillum chlorophenolicum FA1, a pure bacterial strain capable of absorbing heavy metals and degrading polycyclic aromatic hydrocarbons (PAHs), was selected as the representative functional bacterium in this study. A series of batch and column experiments were conducted to investigate the transport and deposition behavior of strain FA1 in saturated porous media. The effects of physical (grain size), chemical (ionic strength, humic acid), and biological factors (living/dead cells) were studied in detail. In addition, numerical simulations of breakthrough curve (BTC) data were also performed for information gathering. Results of this study could advance our understanding of functional bacteria transport and help to develop successful bioremediation strategies. This work was financially supported by the National Natural Science Foundation of China -Xinjiang Project (U1503282), the National Natural Science Foundation of China (41030746, 41102148), and the Natural Science Foundation of Jiangsu Province (BK20151385). Keywords: Herbaspirillum chlorophenolicum FA1, bacteria, porous media, transport, modeling

Pluripotent cell models of fanconi anemia identify the early pathological defect in human hemoangiogenic progenitors.

PubMed

Suzuki, Naoya M; Niwa, Akira; Yabe, Miharu; Hira, Asuka; Okada, Chihiro; Amano, Naoki; Watanabe, Akira; Watanabe, Ken-Ichiro; Heike, Toshio; Takata, Minoru; Nakahata, Tatsutoshi; Saito, Megumu K

2015-04-01

Fanconi anemia (FA) is a disorder of genomic instability characterized by progressive bone marrow failure (BMF), developmental abnormalities, and an increased susceptibility to cancer. Although various consequences in hematopoietic stem/progenitor cells have been attributed to FA-BMF, the quest to identify the initial pathological event is still ongoing. To address this issue, we established induced pluripotent stem cells (iPSCs) from fibroblasts of six patients with FA and FANCA mutations. An improved reprogramming method yielded iPSC-like colonies from all patients, and iPSC clones were propagated from two patients. Quantitative evaluation of the differentiation ability demonstrated that the differentiation propensity toward the hematopoietic and endothelial lineages is already defective in early hemoangiogenic progenitors. The expression levels of critical transcription factors were significantly downregulated in these progenitors. These data indicate that the hematopoietic consequences in FA patients originate from the early hematopoietic stage and highlight the potential usefulness of iPSC technology for elucidating the pathogenesis of FA-BMF. ©AlphaMed Press.

Insights into the effects of polygalacturonase FaPG1 gene silencing on pectin matrix disassembly, enhanced tissue integrity, and firmness in ripe strawberry fruits

PubMed Central

Posé, Sara; Paniagua, Candelas; Cifuentes, Manuel; Blanco-Portales, Rosario; Quesada, Miguel A.; Mercado, José A.

2013-01-01

Antisense-mediated down-regulation of the fruit-specific polygalacturonase (PG) gene FaPG1 in strawberries (Fragaria×ananassa Duch.) has been previously demonstrated to reduce fruit softening and to extend post-harvest shelf life, despite the low PG activity detected in this fruit. The improved fruit traits were suggested to be attributable to a reduced cell wall disassembly due to FaPG1 silencing. This research provides empirical evidence that supports this assumption at the biochemical, cellular, and tissue levels. Cell wall modifications of two independent transgenic antisense lines that demonstrated a >90% reduction in FaPG1 transcript levels were analysed. Sequential extraction of cell wall fractions from control and ripe fruits exhibited a 42% decrease in pectin solubilization in transgenic fruits. A detailed chromatographic analysis of the gel filtration pectin profiles of the different cell wall fractions revealed a diminished depolymerization of the more tightly bound pectins in transgenic fruits, which were solubilized with both a chelating agent and sodium carbonate. The cell wall extracts from antisense FaPG1 fruits also displayed less severe in vitro swelling. A histological analysis revealed more extended cell–cell adhesion areas and an enhanced tissue integrity in transgenic ripe fruits. An immunohistological analysis of fruit sections using the JIM5 antibody against low methyl-esterified pectins demonstrated a higher labelling in transgenic fruit sections, whereas minor differences were observed with JIM7, an antibody that recognizes highly methyl-esterified pectins. These results support that the increased firmness of transgenic antisense FaPG1 strawberry fruits is predominantly due to a decrease in pectin solubilization and depolymerization that correlates with more tightly attached cell wall-bound pectins. This limited disassembly in the transgenic lines indicates that these pectin fractions could play a key role in tissue integrity

Cloning and expression analysis of FaPR-1 gene in strawberry

NASA Astrophysics Data System (ADS)

Mo, Fan; Luo, Ya; Ge, Cong; Mo, Qin; Ling, Yajie; Luo, Shu; Tang, Haoru

2018-04-01

The FaPR-1 gene was cloned by RT-PCR from `Benihoppe' strawberry and its bioinformatics analysis was conducted. The results showed that the open reading frame was 483 bp encoding encoding l60 amino acids which protein molecular weight and theoretical isoelectricity were 17854.17 and 8.72 respectively. Subcellular localization prediction shows that this gene is located extracellularly. By comparing strawberry FaPR-l and other plant Pathogenesis-related protein, homology and phylogenetic tree construction showed that the homology with grapes, peach is relatively close. In the treatments of ABA, sucrose and the mixture of the two, the expression of FaPR-1 in strawberry fruit were significantly increased.

Cytotoxicity of the dicarboximide fungicides, vinclozolin and iprodione, in rat hepatoma-derived Fa32 cells.

PubMed

Dierickx, Paul J

2004-10-01

Dicarboximide fungicides are widely used to control various fungal species. Their primary action is not known, due to a lack of knowledge concerning the mechanism of action of the dicarboximide group. The cytotoxicities of vinclozolin and iprodione in rat hepatoma-derived Fa32 cells were investigated. Cytotoxicity was measured by neutral red uptake inhibition after treatment for 24 hours. Iprodione was more toxic than vinclozolin. Vinclozolin was less toxic in glutathione-depleted cells than in control cells. This was also true for iprodione at lower concentrations, but iprodione became more toxic at higher concentrations. Both the fungicides increased the endogenous glutathione content by 20% after 1 hour. After 24 hours, the glutathione content was doubled by vinclozolin, but was not affected by iprodione. No effect on glutathione S-transferase activity or reactive oxygen species formation could be observed. Cytochrome P450-dependent ethoxyresorufin-O-deethylase and pentoxyresorufin-O-depentylase activities were moderately activated by iprodione and strongly activated by vinclozolin. A glutathione-related cytochrome P450-dependent metabolic attack of vinclozolin and iprodione could be responsible for their cytotoxicity in Fa32 cells. Further research is needed to fully elucidate these (or other) mechanisms.

Stromal interaction molecule 1 (STIM1) silencing inhibits tumor growth and promotes cell cycle arrest and apoptosis in hypopharyngeal carcinoma.

PubMed

Sun, Yuanhao; Cui, Xiaobo; Wang, Jun; Wu, Shuai; Bai, Yunfei; Wang, Yaping; Wang, Boqian; Fang, Jugao

2015-05-01

As an important pathway maintaining the balance of intracellular calcium (Ca(2+)), store-operated Ca(2+) entry (SOCE) is critical for cellular functions. Stromal interaction molecule 1 (STIM1), a key component of SOCE, plays a dual role as an endoplasmic reticulum Ca(2+) receptor and an SOCE exciter. Aberrant expression of STIM1 could be discovered in several human cancer cells. However, the role of STIM1 in regulating human hypopharyngeal carcinoma still remains unclear. Real-time polymerase chain reaction (PCR) was used to detect expression of STIM1 in human hypopharyngeal carcinoma cell line FaDu. STIM1 on FaDu cells was knocked down by lentiviral transduction method. The biological impacts after knocking down of STIM1 on FaDu cells were investigated in vitro and in vivo. The result of real-time PCR showed that STIM1 was expressed in FaDu cells. Lentiviral transduction efficiently downregulated the expression of STIM1 in FaDu cells at both mRNA and protein levels. Significant downregulation of STIM1 on FaDu cells inhibited cell proliferation, induced cell cycle arrest in G0/G1 phase, promoted cell apoptosis, and restrained cell growth rate. The antigrowth effect of STIM1 silencing was also discovered in FaDu hypopharyngeal tumor model. Our findings indicate that STIM1 is likely to become a new therapeutic target for hypopharyngeal carcinoma treatment.

Starmerella camargoi f.a., sp. nov., Starmerella ilheusensis f.a., sp. nov., Starmerella litoralis f.a., sp. nov., Starmerella opuntiae f.a., sp. nov., Starmerella roubikii f.a., sp. nov. and Starmerella vitae f.a., sp. nov., isolated from flowers and bees, and transfer of related Candida species to the genus Starmerella as new combinations.

PubMed

Santos, Ana Raquel O; Leon, Marina P; Barros, Katharina O; Freitas, Larissa F D; Hughes, Alice F S; Morais, Paula B; Lachance, Marc-André; Rosa, Carlos A

2018-04-01

Six novel yeast species, Starmerella camargoi f.a., sp. nov., Starmerella ilheusensis f.a., sp. nov., Starmerella litoralis f.a., Starmerella opuntiae f.a., sp. nov., sp. nov., Starmerella roubikii f.a., sp. nov. and Starmerella vitae f.a, sp. nov. are proposed to accommodate 19 isolates recovered from ephemeral flowers or bees in Brazil, Costa Rica and Belize. Sequence analysis of the ITS-5.8S region (when available) and the D1/D2 domains of the large subunit of the rRNA gene showed that the six novel yeasts are phylogenetically related to several species of the Starmerella clade. The type strains are Starmerella camargoi f.a., sp. nov. UFMG-CM-Y595 T (=CBS 14130 T ; Mycobank number MB 822640), Starmerella ilheusensis f.a., sp. nov. UFMG-CM-Y596 T (=CBS CBS14131 T ; MB 822641), Starmerella litoralis f.a., sp. nov. UFMG-CM-Y603 T (=CBS14104 T ; MB 822642), Starmerella opuntiae f.a., sp. nov. UFMG-CM-Y286 T (=CBS 13466 T ; MB 822643), Starmerella roubikii f.a., sp. nov. UWOPS 01-191.1 (=CBS 15148; MB 822645) and Starmerella vitae f.a., sp. nov. UWOPS 00-107.2 (=CBS 15147 T ; MB 822646). In addition, 25 species currently assigned to the genus Candida are reassigned formally to the genus Starmerella.

The potassium channel FaTPK1 plays a critical role in fruit quality formation in strawberry (Fragaria × ananassa).

PubMed

Wang, Shufang; Song, Miaoyu; Guo, Jiaxuan; Huang, Yun; Zhang, Fangfang; Xu, Cheng; Xiao, Yinghui; Zhang, Lusheng

2018-03-01

Potassium (K + ), an abundant cation in plant cells, is important in fruit development and plant resistance. However, how cellular K + is directed by potassium channels in fruit development and quality formation of strawberry (Fragaria × ananassa) is not yet fully clear. Here, a two-pore K + (TPK) channel gene in strawberry, FaTPK1, was cloned using reverse transcription-PCR. A green fluorescent protein subcellular localization analysis showed that FaTPK1 localized in the vacuole membrane. A transcription analysis indicated that the mRNA expression level of FaTPK1 increased rapidly and was maintained at a high level in ripened fruit, which was coupled with the fruit's red colour development, suggesting that FaTPK1 is related to fruit quality formation. The down- and up-regulation of the FaTPK1 mRNA expression levels using RNA interference and overexpression, respectively, inhibited and promoted fruit ripening, respectively, as demonstrated by consistent changes in firmness and the contents of soluble sugars, anthocyanin and abscisic acid, as well as the transcript levels of ripening-regulated genes PG1 (polygalacturonase), GAL6 (beta-galactosidase), XYL2 (D-xylulose reductase), SUT1 (sucrose transporter), CHS (chalcone synthase) and CHI (chalcone flavanone isomerase). Additionally, the regulatory changes influenced fruit resistance to Botrytis cinerea. An isothermal calorimetry analysis showed that the Escherichia coli-expressed FaTPK1 recombinant protein could bind K + with a binding constant of 2.1 × 10 -3  m -1 and a dissociation constant of 476 μm. Thus, the strawberry TPK1 is a ubiquitously expressed, tonoplast-localized two-pore potassium channel that plays important roles in fruit ripening and quality formation. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

A case report and literature review of Fanconi Anemia (FA) diagnosed by genetic testing.

PubMed

Solomon, Ponnumony John; Margaret, Priya; Rajendran, Ramya; Ramalingam, Revathy; Menezes, Godfred A; Shirley, Alph S; Lee, Seung Jun; Seong, Moon-Woo; Park, Sung Sup; Seol, Dodam; Seo, Soo Hyun

2015-05-08

Fanconi anemia (FA) is a genetically heterogeneous rare autosomal recessive disorder characterized by congenital malformations, hematological problems and predisposition to malignancies. The genes that have been found to be mutated in FA patients are called FANC. To date 16 distinct FANC genes have been reported. Among these, mutations in FANCA are the most frequent among FA patients worldwide which account for 60- 65%. In this study, a nine years old male child was brought to our hospital one year ago for opinion and advice. He was the third child born to consanguineous parents. The mutation analyses were performed for proband, parents, elder sibling and the relatives [maternal aunt and maternal aunt's son (cousin)]. Molecular genetic testing [targeted next-generation sequencing (MiSeq, Illumina method)] was performed by mutation analysis in 15 genes involved. Entire coding exons and their flanking regions of the genes were analysed. Sanger sequencing [(ABI 3730 analyzer by Applied Biosystems)] was performed using primers specific for 43 coding exons of the FANCA gene. A novel splice site mutation, c.3066 + 1G > T, (IVS31 + 1G > T), homozygote was detected by sequencing in the patient. The above sequence variant was identified in heterozygous state in his parents. Further, the above sequence variant was not identified in other family members (elder sibling, maternal aunt and cousin). It is concluded that genetic study should be done if possible in all the cases of suspected FA, including siblings, parents and close blood relatives. It will help us to plan appropriate treatment and also to select suitable donor for hematopoietic stem cell transplantation and to plan for genetic counseling. In addition to the case report, the main focus of this manuscript was to review literature on role of FANCA gene in FA since large number of FANCA mutations and polymorphisms have been identified.

Targeting property and toxicity of a novel ultrasound contrast agent microbubble carrying the targeting and drug-loaded complex FA-CNTs-PTX on MCF7 cells.

PubMed

Zhang, Jie; Zhang, Yu; Liu, Junxi; Li, Guozhong; Wen, Zhaohui; Zhao, Yue; Zhang, Xiangyu; Liu, Fenghua

2017-10-01

The application of ultrasound contrast agents not only is confined to the enhancement of ultrasound imaging but also has started to be used as a drug system for diagnosis and treatment. In this paper, Span60 and PEG1500 were used as membrane materials, and a new targeting and drug-loading multifunctional ultrasound contrast agent microbubble enveloping the FA-CNTs-PTX complex was successfully prepared by acoustic cavitation. With the breast cancer cell line MCF7 as the research target, the effects of the microbubble with FA-CNTs-PTX on the proliferation and toxicity of MCF7 cells were studied using a CCK-8 and AO/EB double-staining method. The influences of the microbubbles with FA-CNTs-PTX on the cellular morphology and apoptosis period of the MCF7 cells were detected using an inverted fluorescence microscope. The apoptosis of MCF7 cells induced by the microbubbles with FA-CNTs-PTX was investigated with flow cytometry and an annexin and PI double staining fluorescence quantitative analysis. The results indicated that the ultrasound contrast agent microbubble with FA-CNTs-PTX remarkably inhibited the proliferation of MCF7 cells, which was mainly controlled by the drug loading rate and the nanometer size of the microbubbles. Moreover, the proliferative inhibition rate of the microbubbles with FA-CNTs-PTX was related to the cell apoptosis period of MCF7 cells. Its inhibition degree on the proliferation of MCF7 cells was higher than that of the hepatoma HepG2 cells. The apoptosis rate of MCF7 cells induced by the microbubbles with FA-CNTs-PTX was higher than that of normal human umbilical vein endothelial cells (HUVECs), and the microbubbles with FA-CNTs-PTX could target the MCF7 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Mass spectrometry of the lithium adducts of diacylglycerols containing hydroxy FA in castor oil and two normal FA

USDA-ARS?s Scientific Manuscript database

Castor oil can be used in industry. The molecular species of triacylglycerols containing hydroxy fatty acids (FA) in castor oil have been identified. We report here the identification of twelve diacylglycerols (DAG) containing hydroxy FA in castor oil using positive ion electrospray ionization mass ...

Construct validity of the Heart Failure Screening Tool (Heart-FaST) to identify heart failure patients at risk of poor self-care: Rasch analysis.

PubMed

Reynolds, Nicholas A; Ski, Chantal F; McEvedy, Samantha M; Thompson, David R; Cameron, Jan

2018-02-14

The aim of this study was to psychometrically evaluate the Heart Failure Screening Tool (Heart-FaST) via: (1) examination of internal construct validity; (2) testing of scale function in accordance with design; and (3) recommendation for change/s, if items are not well adjusted, to improve psychometric credential. Self-care is vital to the management of heart failure. The Heart-FaST may provide a prospective assessment of risk, regarding the likelihood that patients with heart failure will engage in self-care. Psychometric validation of the Heart-FaST using Rasch analysis. The Heart-FaST was administered to 135 patients (median age = 68, IQR = 59-78 years; 105 males) enrolled in a multidisciplinary heart failure management program. The Heart-FaST is a nurse-administered tool for screening patients with HF at risk of poor self-care. A Rasch analysis of responses was conducted which tested data against Rasch model expectations, including whether items serve as unbiased, non-redundant indicators of risk and measure a single construct and that rating scales operate as intended. The results showed that data met Rasch model expectations after rescoring or deleting items due to poor discrimination, disordered thresholds, differential item functioning, or response dependence. There was no evidence of multidimensionality which supports the use of total scores from Heart-FaST as indicators of risk. Aggregate scores from this modified screening tool rank heart failure patients according to their "risk of poor self-care" demonstrating that the Heart-FaST items constitute a meaningful scale to identify heart failure patients at risk of poor engagement in heart failure self-care. © 2018 John Wiley & Sons Ltd.

pH-sensitive Au–BSA–DOX–FA nanocomposites for combined CT imaging and targeted drug delivery

PubMed Central

Huang, He; Yang, Da-Peng; Liu, Minghuan; Wang, Xiangsheng; Zhang, Zhiyong; Zhou, Guangdong; Liu, Wei; Cao, Yilin; Zhang, Wen Jie; Wang, Xiansong

2017-01-01

Albumin-based nanoparticles (NPs) as a drug delivery system have attracted much attention owing to their nontoxicity, non-immunogenicity, great stability and ability to bind to many therapeutic drugs. Herein, bovine serum albumin (BSA) was utilized as a template to prepare Au–BSA core/shell NPs. The outer layer BSA was subsequently conjugated with cis-aconityl doxorubicin (DOX) and folic acid (FA) to create Au–BSA–DOX–FA nanocomposites. A list of characterizations was undertaken to identify the successful conjugation of drug molecules and targeted agents. In vitro cytotoxicity using a cell counting kit-8 (CCK-8) assay indicated that Au–BSA NPs did not display obvious cytotoxicity to MGC-803 and GES-1 cells in the concentration range of 0–100 μg/mL, which can therefore be used as a safe drug delivery carrier. Furthermore, compared with free DOX, Au–BSA–DOX–FA nanocomposites exhibited a pH-sensitive drug release ability and superior antitumor activity in a drug concentration-dependent manner. In vivo computed tomography (CT) imaging experiments showed that Au–BSA–DOX–FA nanocomposites could be used as an efficient and durable CT contrast agent for targeted CT imaging of the folate receptor (FR) overexpressed in cancer tissues. In vivo antitumor experiments demonstrated that Au–BSA–DOX–FA nanocomposites have selective antitumor activity effects on FR-overexpressing tumors and no adverse effects on normal tissues and organs. In conclusion, the Au–BSA–DOX–FA nanocomposite exhibits selective targeting activity, X-ray attenuation activity and pH-sensitive drug release activity. Therefore, it can enhance CT imaging and improve the targeting therapeutic efficacy of FR-overexpressing gastric cancers. Our findings suggest that Au–BSA–DOX–FA nanocomposite is a novel drug delivery carrier and a promising candidate for cancer theranostic applications. PMID:28435261

The carboxyl terminus of FANCE recruits FANCD2 to the Fanconi Anemia (FA) E3 ligase complex to promote the FA DNA repair pathway.

PubMed

Polito, David; Cukras, Scott; Wang, Xiaozhe; Spence, Paige; Moreau, Lisa; D'Andrea, Alan D; Kee, Younghoon

2014-03-07

Fanconi anemia (FA) is a genome instability syndrome characterized by bone marrow failure and cellular hypersensitivity to DNA cross-linking agents. In response to DNA damage, the FA pathway is activated through the cooperation of 16 FA proteins. A central player in the pathway is a multisubunit E3 ubiquitin ligase complex or the FA core complex, which monoubiquitinates its substrates FANCD2 and FANCI. FANCE, a subunit of the FA core complex, plays an essential role by promoting the integrity of the complex and by directly recognizing FANCD2. To delineate its role in substrate ubiquitination from the core complex assembly, we analyzed a series of mutations within FANCE. We report that a phenylalanine located at the highly conserved extreme C terminus, referred to as Phe-522, is a critical residue for mediating the monoubiquitination of the FANCD2-FANCI complex. Using the FANCE mutant that specifically disrupts the FANCE-FANCD2 interaction as a tool, we found that the interaction-deficient mutant conferred cellular sensitivity in reconstituted FANCE-deficient cells to a similar degree as FANCE null cells, suggesting the significance of the FANCE-FANCD2 interaction in promoting cisplatin resistance. Intriguingly, ectopic expression of the FANCE C terminus fragment alone in FA normal cells disrupts DNA repair, consolidating the importance of the FANCE-FANCD2 interaction in the DNA cross-link repair.

The Carboxyl Terminus of FANCE Recruits FANCD2 to the Fanconi Anemia (FA) E3 Ligase Complex to Promote the FA DNA Repair Pathway*

PubMed Central

Polito, David; Cukras, Scott; Wang, Xiaozhe; Spence, Paige; Moreau, Lisa; D'Andrea, Alan D.; Kee, Younghoon

2014-01-01

Fanconi anemia (FA) is a genome instability syndrome characterized by bone marrow failure and cellular hypersensitivity to DNA cross-linking agents. In response to DNA damage, the FA pathway is activated through the cooperation of 16 FA proteins. A central player in the pathway is a multisubunit E3 ubiquitin ligase complex or the FA core complex, which monoubiquitinates its substrates FANCD2 and FANCI. FANCE, a subunit of the FA core complex, plays an essential role by promoting the integrity of the complex and by directly recognizing FANCD2. To delineate its role in substrate ubiquitination from the core complex assembly, we analyzed a series of mutations within FANCE. We report that a phenylalanine located at the highly conserved extreme C terminus, referred to as Phe-522, is a critical residue for mediating the monoubiquitination of the FANCD2-FANCI complex. Using the FANCE mutant that specifically disrupts the FANCE-FANCD2 interaction as a tool, we found that the interaction-deficient mutant conferred cellular sensitivity in reconstituted FANCE-deficient cells to a similar degree as FANCE null cells, suggesting the significance of the FANCE-FANCD2 interaction in promoting cisplatin resistance. Intriguingly, ectopic expression of the FANCE C terminus fragment alone in FA normal cells disrupts DNA repair, consolidating the importance of the FANCE-FANCD2 interaction in the DNA cross-link repair. PMID:24451376

Poly(l-lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA): a bioactive copolymer for specific targeting to folate receptor-positive cancer cells.

PubMed

Chen, Yin; Cao, Wenbin; Zhou, Junli; Pidhatika, Bidhari; Xiong, Bin; Huang, Lu; Tian, Qian; Shu, Yiwei; Wen, Weijia; Hsing, I-Ming; Wu, Hongkai

2015-02-04

In this study, we present the preparation, characterization and application of a novel bioactive copolymer poly(l-lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA), which has a specific interaction with folate receptor (FR)-positive cancer cells. Glass surface immobilized with PLL-g-PMOXA-c-FA was demonstrated to be adhesive to FR-positive cancer cells (HeLa, JEG-3) while nonadhesive to FR-negative ones (MCF-7, HepG2) in 3 h. The specific interaction between conjugated FA on the substrate and FRs on the cells could hardly be inhibited unless a high concentration (5 mM) of free FA was used due to the multivalent nature of it. The FA functionality ratio of the copolymer on the substrate had a significant influence on the adhesion of HeLa cells, and our experiments revealed that the affinity of the substrate to the cells declined dramatically with the decrease of functionality ratio. This was believed to be caused by the polydispersity of PMOXA tethers, as supported by GPC and ToF-SIMS data. As a proof of concept in the application of our material, we demonstrated successful recovery of HeLa cells from mixture with MCF-7 (1:100) on the copolymer-coated glass, and our results showed that both high sensitivity (95.6 ± 13.3%) and specificity (24.3 ± 8.6%) were achieved.

Co-expression analysis identifies CRC and AP1 the regulator of Arabidopsis fatty acid biosynthesis.

PubMed

Han, Xinxin; Yin, Linlin; Xue, Hongwei

2012-07-01

Fatty acids (FAs) play crucial rules in signal transduction and plant development, however, the regulation of FA metabolism is still poorly understood. To study the relevant regulatory network, fifty-eight FA biosynthesis genes including de novo synthases, desaturases and elongases were selected as "guide genes" to construct the co-expression network. Calculation of the correlation between all Arabidopsis thaliana (L.) genes with each guide gene by Arabidopsis co-expression dating mining tools (ACT) identifies 797 candidate FA-correlated genes. Gene ontology (GO) analysis of these co-expressed genes showed they are tightly correlated to photosynthesis and carbohydrate metabolism, and function in many processes. Interestingly, 63 transcription factors (TFs) were identified as candidate FA biosynthesis regulators and 8 TF families are enriched. Two TF genes, CRC and AP1, both correlating with 8 FA guide genes, were further characterized. Analyses of the ap1 and crc mutant showed the altered total FA composition of mature seeds. The contents of palmitoleic acid, stearic acid, arachidic acid and eicosadienoic acid are decreased, whereas that of oleic acid is increased in ap1 and crc seeds, which is consistent with the qRT-PCR analysis revealing the suppressed expression of the corresponding guide genes. In addition, yeast one-hybrid analysis and electrophoretic mobility shift assay (EMSA) revealed that CRC can bind to the promoter regions of KCS7 and KCS15, indicating that CRC may directly regulate FA biosynthesis. © 2012 Institute of Botany, Chinese Academy of Sciences.

Fitness costs and stability of Cry1Fa resistance in Brazilian populations of Spodoptera frugiperda.

PubMed

Santos-Amaya, Oscar F; Tavares, Clébson S; Rodrigues, João Victor C; Campos, Silverio O; Guedes, Raul Narciso C; Alves, Analiza P; Pereira, Eliseu José G

2017-01-01

The presence of fitness costs of resistance to Bacillus thuringiensis (Bt) insecticidal proteins in insect populations may delay or even reverse the local selection of insect resistance to Bt transgenic crops, and deserves rigorous investigation. Here we assessed the fitness costs associated with Cry1Fa resistance in two strains of fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), derived from field collections in different Brazilian regions and further selected in the laboratory for high levels of resistance to Cry1Fa using leaves of TC1507 corn. Fitness components were compared using paired resistant and susceptible strains with similar genetic backgrounds and F 1 generations from reciprocal crosses, all of them reared on non-transgenic corn leaves. No apparent life history costs in the larval stage were observed in the Bt-resistant strains. Moreover, the resistance remained stable for seven generations in the absence of selection, with no decrease in the proportion of resistant individuals. Larval respiration rates were also similar between resistant and susceptible homozygotes, and heterozygotes displayed respiration rates and demographic performance equal or superior to those of susceptible homozygotes. In combination, these results indicate the lack of strong fitness costs associated with resistance to Cry1Fa in the fall armyworm strains studied. These findings suggest that Cry1Fa resistance in S. frugiperda populations is unlikely to be counterselected in Cry1Fa-free environments. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Diabetes-associated microbiota in fa/fa rats is modified by Roux-en-Y gastric bypass.

PubMed

Arora, Tulika; Seyfried, Florian; Docherty, Neil G; Tremaroli, Valentina; le Roux, Carel W; Perkins, Rosie; Bäckhed, Fredrik

2017-09-01

Roux-en-Y gastric bypass (RYGB) and duodenal jejunal bypass (DJB), two different forms of bariatric surgery, are associated with improved glucose tolerance, but it is not clear whether the gut microbiota contributes to this effect. Here we used fa/fa rats as a model of impaired glucose tolerance to investigate whether (i) the microbiota varies between fa/fa and nondiabetic fa/+ rats; (ii) the microbiota of fa/fa rats is affected by RYGB and/or DJB; and (iii) surgically induced microbiota alterations contribute to glucose metabolism. We observed a profound expansion of Firmicutes (specifically, Lactobacillus animalis and Lactobacillus reuteri) in the small intestine of diabetic fa/fa compared with nondiabetic fa/+ rats. RYGB-, but not DJB-, treated fa/fa rats exhibited greater microbiota diversity in the ileum and lower L. animalis and L. reuteri abundance compared with sham-operated fa/fa rats in all intestinal segments, and their microbiota composition resembled that of unoperated fa/+ rats. To investigate the functional role of RYGB-associated microbiota alterations, we transferred microbiota from sham- and RYGB-treated fa/fa rats to germ-free mice. The metabolic phenotype of RYGB-treated rats was not transferred by the transplant of ileal microbiota. In contrast, postprandial peak glucose levels were lower in mice that received cecal microbiota from RYGB- versus sham-operated rats. Thus, diabetes-associated microbiota alterations in fa/fa rats can be modified by RYGB, and modifications in the cecal microbiota may partially contribute to improved glucose tolerance after RYGB.

Diabetes-associated microbiota in fa/fa rats is modified by Roux-en-Y gastric bypass

PubMed Central

Arora, Tulika; Seyfried, Florian; Docherty, Neil G; Tremaroli, Valentina; le Roux, Carel W; Perkins, Rosie; Bäckhed, Fredrik

2017-01-01

Roux-en-Y gastric bypass (RYGB) and duodenal jejunal bypass (DJB), two different forms of bariatric surgery, are associated with improved glucose tolerance, but it is not clear whether the gut microbiota contributes to this effect. Here we used fa/fa rats as a model of impaired glucose tolerance to investigate whether (i) the microbiota varies between fa/fa and nondiabetic fa/+ rats; (ii) the microbiota of fa/fa rats is affected by RYGB and/or DJB; and (iii) surgically induced microbiota alterations contribute to glucose metabolism. We observed a profound expansion of Firmicutes (specifically, Lactobacillus animalis and Lactobacillus reuteri) in the small intestine of diabetic fa/fa compared with nondiabetic fa/+ rats. RYGB-, but not DJB-, treated fa/fa rats exhibited greater microbiota diversity in the ileum and lower L. animalis and L. reuteri abundance compared with sham-operated fa/fa rats in all intestinal segments, and their microbiota composition resembled that of unoperated fa/+ rats. To investigate the functional role of RYGB-associated microbiota alterations, we transferred microbiota from sham- and RYGB-treated fa/fa rats to germ-free mice. The metabolic phenotype of RYGB-treated rats was not transferred by the transplant of ileal microbiota. In contrast, postprandial peak glucose levels were lower in mice that received cecal microbiota from RYGB- versus sham-operated rats. Thus, diabetes-associated microbiota alterations in fa/fa rats can be modified by RYGB, and modifications in the cecal microbiota may partially contribute to improved glucose tolerance after RYGB. PMID:28524868

Whole exome sequencing reveals concomitant mutations of multiple FA genes in individual Fanconi anemia patients

PubMed Central

2014-01-01

Background Fanconi anemia (FA) is a rare inherited genetic syndrome with highly variable clinical manifestations. Fifteen genetic subtypes of FA have been identified. Traditional complementation tests for grouping studies have been used generally in FA patients and in stepwise methods to identify the FA type, which can result in incomplete genetic information from FA patients. Methods We diagnosed five pediatric patients with FA based on clinical manifestations, and we performed exome sequencing of peripheral blood specimens from these patients and their family members. The related sequencing data were then analyzed by bioinformatics, and the FANC gene mutations identified by exome sequencing were confirmed by PCR re-sequencing. Results Homozygous and compound heterozygous mutations of FANC genes were identified in all of the patients. The FA subtypes of the patients included FANCA, FANCM and FANCD2. Interestingly, four FA patients harbored multiple mutations in at least two FA genes, and some of these mutations have not been previously reported. These patients’ clinical manifestations were vastly different from each other, as were their treatment responses to androstanazol and prednisone. This finding suggests that heterozygous mutation(s) in FA genes could also have diverse biological and/or pathophysiological effects on FA patients or FA gene carriers. Interestingly, we were not able to identify de novo mutations in the genes implicated in DNA repair pathways when the sequencing data of patients were compared with those of their parents. Conclusions Our results indicate that Chinese FA patients and carriers might have higher and more complex mutation rates in FANC genes than have been conventionally recognized. Testing of the fifteen FANC genes in FA patients and their family members should be a regular clinical practice to determine the optimal care for the individual patient, to counsel the family and to obtain a better understanding of FA pathophysiology

Whole exome sequencing reveals concomitant mutations of multiple FA genes in individual Fanconi anemia patients.

PubMed

Chang, Lixian; Yuan, Weiping; Zeng, Huimin; Zhou, Quanquan; Wei, Wei; Zhou, Jianfeng; Li, Miaomiao; Wang, Xiaomin; Xu, Mingjiang; Yang, Fengchun; Yang, Yungui; Cheng, Tao; Zhu, Xiaofan

2014-05-15

Fanconi anemia (FA) is a rare inherited genetic syndrome with highly variable clinical manifestations. Fifteen genetic subtypes of FA have been identified. Traditional complementation tests for grouping studies have been used generally in FA patients and in stepwise methods to identify the FA type, which can result in incomplete genetic information from FA patients. We diagnosed five pediatric patients with FA based on clinical manifestations, and we performed exome sequencing of peripheral blood specimens from these patients and their family members. The related sequencing data were then analyzed by bioinformatics, and the FANC gene mutations identified by exome sequencing were confirmed by PCR re-sequencing. Homozygous and compound heterozygous mutations of FANC genes were identified in all of the patients. The FA subtypes of the patients included FANCA, FANCM and FANCD2. Interestingly, four FA patients harbored multiple mutations in at least two FA genes, and some of these mutations have not been previously reported. These patients' clinical manifestations were vastly different from each other, as were their treatment responses to androstanazol and prednisone. This finding suggests that heterozygous mutation(s) in FA genes could also have diverse biological and/or pathophysiological effects on FA patients or FA gene carriers. Interestingly, we were not able to identify de novo mutations in the genes implicated in DNA repair pathways when the sequencing data of patients were compared with those of their parents. Our results indicate that Chinese FA patients and carriers might have higher and more complex mutation rates in FANC genes than have been conventionally recognized. Testing of the fifteen FANC genes in FA patients and their family members should be a regular clinical practice to determine the optimal care for the individual patient, to counsel the family and to obtain a better understanding of FA pathophysiology.

Middle region of FancM interacts with Mhf and Rmi1 in silkworms, a species lacking the Fanconi anaemia (FA) core complex.

PubMed

Sugahara, R; Mon, H; Lee, J M; Kusakabe, T

2014-04-01

The Fanconi anaemia (FA) pathway is responsible for interstrand crosslink (ICL) repair. Among the FA core complex components, FANCM is believed to act as a damage sensor for the ICL-blocked replication fork and also as a molecular platform for FA core complex assembly and interaction with Bloom's syndrome (BS) complex that is thought to play an important role in the processing of DNA structures such as stalled replication forks. In the present study, we found that in silkworms, Bombyx mori, a species lacking the major FA core complex components (FANCA, B, C, E, F, and G), FancM is required for FancD2 monoubiquitination and cell proliferation in the presence of mitomycin C (MMC). Silkworm FancM (BmFancM) was phosphorylated in the middle regions, and the modification was associated with its subcellular localization. In addition, BmFancM interacted with Mhf1, a histone-fold protein, and Rmi1, a subunit of the BS complex, in the different regions. The interaction region containing at least these two protein-binding domains played an essential role in FancM-dependent resistance to MMC. Our results suggest that BmFancM also acts as a platform for recruitment of both the FA protein and the BS protein, although the silkworm genome seems to lose FAAP24, a FancM-binding partner protein in mammals. © 2013 The Royal Entomological Society.

The FA pathway counteracts oxidative stress through selective protection of antioxidant defense gene promoters.

PubMed

Du, Wei; Rani, Reena; Sipple, Jared; Schick, Jonathan; Myers, Kasiani C; Mehta, Parinda; Andreassen, Paul R; Davies, Stella M; Pang, Qishen

2012-05-03

Oxidative stress has been implicated in the pathogenesis of many human diseases including Fanconi anemia (FA), a genetic disorder associated with BM failure and cancer. Here we show that major antioxidant defense genes are down-regulated in FA patients, and that gene down-regulation is selectively associated with increased oxidative DNA damage in the promoters of the antioxidant defense genes. Assessment of promoter activity and DNA damage repair kinetics shows that increased initial damage, rather than a reduced repair rate, contributes to the augmented oxidative DNA damage. Mechanistically, FA proteins act in concert with the chromatin-remodeling factor BRG1 to protect the promoters of antioxidant defense genes from oxidative damage. Specifically, BRG1 binds to the promoters of the antioxidant defense genes at steady state. On challenge with oxidative stress, FA proteins are recruited to promoter DNA, which correlates with significant increase in the binding of BRG1 within promoter regions. In addition, oxidative stress-induced FANCD2 ubiquitination is required for the formation of a FA-BRG1-promoter complex. Taken together, these data identify a role for the FA pathway in cellular antioxidant defense.

Time to Detection with BacT/Alert FA Plus Compared to BacT/Alert FA Blood Culture Media.

PubMed

Nutman, A; Fisher Even-Tsur, S; Shapiro, G; Braun, T; Schwartz, D; Carmeli, Y

2016-09-01

Rapid identification of the causative pathogen in patients with bacteremia allows adjustment of antibiotic therapy and improves patient outcomes. We compared in vitro and real-life time to detection (TTD) of two blood culture media, BacT/Alert FA (FA) and BacT/Alert FA Plus (FA Plus), for the nine most common species of bacterial pathogens recovered from blood samples. Experimental data from simulated cultures was compared with microbiology records of TTD for both culture media with growth of the species of interest in clinical blood cultures. In the experimental conditions, median TTD was 3.8 hours (23.9 %) shorter using FA Plus media. The magnitude of reduction differed between species. Similarly, in real life data, FA Plus had shorter TTD than FA media; however, the difference between culture media was smaller, and median TTD was only 1 hour (8.5 %) less. We found shorter TTD with BacT/Alert FA Plus culture media, both experimentally and in real-life conditions and unrelated to antibiotic neutralization, highlighting the importance of appropriate blood culture media selection.

Fe3O4@mSiO2-FA-CuS-PEG nanocomposites for magnetic resonance imaging and targeted chemo-photothermal synergistic therapy of cancer cells.

PubMed

Gao, Zhifang; Liu, Xijian; Deng, Guoying; Zhou, Feng; Zhang, Lijuan; Wang, Qian; Lu, Jie

2016-09-14

In this work, a new multifunctional nanoplatform (Fe3O4@mSiO2-FA-CuS-PEG nanocomposite) for magnetic resonance imaging (MRI) and targeted chemo-photothermal therapy, was firstly fabricated on the basis of magnetic mesoporous silica nanoparticles (Fe3O4@mSiO2), on which folic acid (FA) was grafted as the targeting reagent, CuS nanocrystals were attached as the photothermal agent, and polyethylene glycol (PEG) was coupled to improve biocompatibility. The characterization results demonstrated that the fabricated Fe3O4@mSiO2-FA-CuS-PEG nanocomposites not only showed strong magnetism and excellent MRI performance, but also had a high doxorubicin (DOX, an anticancer drug) loading capacity (22.1%). The loaded DOX can be sustainably released, which was apt to be controlled by pH adjustment and near infrared (NIR) laser irradiation. More importantly, targeted delivery of the DOX-loaded Fe3O4@mSiO2-FA-CuS-PEG nanocomposites could be accomplished in HeLa cells via the receptor-mediated endocytosis pathway, and this exhibited synergistic effect of chemotherapy and photothermal therapy against HeLa cells under irradiation with a 915 nm laser. Therefore, the fabricated multifunctional Fe3O4@mSiO2-FA-CuS-PEG nanocomposite has a great potential in image-guided therapy of cancers.

FAAP20: a novel ubiquitin-binding FA nuclear core-complex protein required for functional integrity of the FA-BRCA DNA repair pathway

PubMed Central

Ali, Abdullah Mahmood; Pradhan, Arun; Singh, Thiyam Ramsingh; Du, Changhu; Li, Jie; Wahengbam, Kebola; Grassman, Elke; Auerbach, Arleen D.; Pang, Qishen

2012-01-01

Fanconi anemia (FA) nuclear core complex is a multiprotein complex required for the functional integrity of the FA-BRCA pathway regulating DNA repair. This pathway is inactivated in FA, a devastating genetic disease, which leads to hematologic defects and cancer in patients. Here we report the isolation and characterization of a novel 20-kDa FANCA-associated protein (FAAP20). We show that FAAP20 is an integral component of the FA nuclear core complex. We identify a region on FANCA that physically interacts with FAAP20, and show that FANCA regulates stability of this protein. FAAP20 contains a conserved ubiquitin-binding zinc-finger domain (UBZ), and binds K-63–linked ubiquitin chains in vitro. The FAAP20-UBZ domain is not required for interaction with FANCA, but is required for DNA-damage–induced chromatin loading of FANCA and the functional integrity of the FA pathway. These findings reveal critical roles for FAAP20 in the FA-BRCA pathway of DNA damage repair and genome maintenance. PMID:22343915

Fanconi Anemia Mesenchymal Stromal Cells-Derived Glycerophospholipids Skew Hematopoietic Stem Cell Differentiation Through Toll-Like Receptor Signaling.

PubMed

Amarachintha, Surya; Sertorio, Mathieu; Wilson, Andrew; Li, Xiaoli; Pang, Qishen

2015-11-01

Fanconi anemia (FA) patients develop bone marrow (BM) failure or leukemia. One standard care for these devastating complications is hematopoietic stem cell transplantation. We identified a group of mesenchymal stromal cells (MSCs)-derived metabolites, glycerophospholipids, and their endogenous inhibitor, 5-(tetradecyloxy)-2-furoic acid (TOFA), as regulators of donor hematopoietic stem and progenitor cells. We provided two pieces of evidence that TOFA could improve hematopoiesis-supporting function of FA MSCs: (a) limiting-dilution cobblestone area-forming cell assay revealed that TOFA significantly increased cobblestone colonies in Fanca-/- or Fancd2-/- cocultures compared to untreated cocultures. (b) Competitive repopulating assay using output cells collected from cocultures showed that TOFA greatly alleviated the abnormal expansion of the donor myeloid (CD45.2+Gr1+Mac1+) compartment in both peripheral blood and BM of recipient mice transplanted with cells from Fanca-/- or Fancd2-/- cocultures. Furthermore, mechanistic studies identified Tlr4 signaling as the responsible pathway mediating the effect of glycerophospholipids. Thus, targeting glycerophospholipid biosynthesis in FA MSCs could be a therapeutic strategy to improve hematopoiesis and stem cell transplantation. © 2015 AlphaMed Press.

The chemokine receptor CCR1 is identified in mast cell-derived exosomes

PubMed Central

Liang, Yuting; Qiao, Longwei; Peng, Xia; Cui, Zelin; Yin, Yue; Liao, Huanjin; Jiang, Min; Li, Li

2018-01-01

Mast cells are important effector cells of the immune system, and mast cell-derived exosomes carrying RNAs play a role in immune regulation. However, the molecular function of mast cell-derived exosomes is currently unknown, and here, we identify differentially expressed genes (DEGs) in mast cells and exosomes. We isolated mast cells derived exosomes through differential centrifugation and screened the DEGs from mast cell-derived exosomes, using the GSE25330 array dataset downloaded from the Gene Expression Omnibus database. Biochemical pathways were analyzed by Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway on the online tool DAVID. DEGs-associated protein-protein interaction networks (PPIs) were constructed using the STRING database and Cytoscape software. The genes identified from these bioinformatics analyses were verified by qRT-PCR and Western blot in mast cells and exosomes. We identified 2121 DEGs (843 up and 1278 down-regulated genes) in HMC-1 cell-derived exosomes and HMC-1 cells. The up-regulated DEGs were classified into two significant modules. The chemokine receptor CCR1 was screened as a hub gene and enriched in cytokine-mediated signaling pathway in module one. Seven genes, including CCR1, CD9, KIT, TGFBR1, TLR9, TPSAB1 and TPSB2 were screened and validated through qRT-PCR analysis. We have achieved a comprehensive view of the pivotal genes and pathways in mast cells and exosomes and identified CCR1 as a hub gene in mast cell-derived exosomes. Our results provide novel clues with respect to the biological processes through which mast cell-derived exosomes modulate immune responses. PMID:29511430

The chemokine receptor CCR1 is identified in mast cell-derived exosomes.

PubMed

Liang, Yuting; Qiao, Longwei; Peng, Xia; Cui, Zelin; Yin, Yue; Liao, Huanjin; Jiang, Min; Li, Li

2018-01-01

Mast cells are important effector cells of the immune system, and mast cell-derived exosomes carrying RNAs play a role in immune regulation. However, the molecular function of mast cell-derived exosomes is currently unknown, and here, we identify differentially expressed genes (DEGs) in mast cells and exosomes. We isolated mast cells derived exosomes through differential centrifugation and screened the DEGs from mast cell-derived exosomes, using the GSE25330 array dataset downloaded from the Gene Expression Omnibus database. Biochemical pathways were analyzed by Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway on the online tool DAVID. DEGs-associated protein-protein interaction networks (PPIs) were constructed using the STRING database and Cytoscape software. The genes identified from these bioinformatics analyses were verified by qRT-PCR and Western blot in mast cells and exosomes. We identified 2121 DEGs (843 up and 1278 down-regulated genes) in HMC-1 cell-derived exosomes and HMC-1 cells. The up-regulated DEGs were classified into two significant modules. The chemokine receptor CCR1 was screened as a hub gene and enriched in cytokine-mediated signaling pathway in module one. Seven genes, including CCR1, CD9, KIT, TGFBR1, TLR9, TPSAB1 and TPSB2 were screened and validated through qRT-PCR analysis. We have achieved a comprehensive view of the pivotal genes and pathways in mast cells and exosomes and identified CCR1 as a hub gene in mast cell-derived exosomes. Our results provide novel clues with respect to the biological processes through which mast cell-derived exosomes modulate immune responses.

The polygalacturonase FaPG1 gene plays a key role in strawberry fruit softening

PubMed Central

García-Gago, Juan A; Posé, Sara; Muñoz-Blanco, Juan; Quesada, Miguel A

2009-01-01

The loss of firm texture is one of the most characteristic physiological processes that occur during the ripening of fleshy fruits. It is generally accepted that the disassembly of primary cell wall and middle lamella is the main factor involved in fruit softening. In this process, polygalacturonase (PG) has been implicated in the degradation of the polyuronide network in several fruits. However, the minor effect of PG downregulation on tomato softening, reported during the nineties, minimized the role of this enzyme in softening. Further works in other fruits are challenging this general assumption, as is occurring in strawberry. The strawberry (Fragaria × ananassa) fruit undergoes an extensive and fast softening that limit its shelf life and postharvest. Traditionally, it has also been considered that PG plays a minor role on this process, due to the low PG activity found in ripened strawberry fruits. Transgenic strawberry plants expressing an antisense sequence of the ripening-specific PG gene FaPG1 have been generated to get an insight into the role of this gene in softening. Half of the transgenic lines analyzed yielded fruits significantly firmer than control, without being affected other fruit parameters such as weight, color or soluble solids. The increase on firmness was maintained after several days of posharvest. In these firmer lines, FaPG1 was silenced to 95%, but total PG activity was only minor reduced. At the cell wall level, transgenic fruits contained a higher amount of covalently bound pectins whereas the soluble fraction was diminished. A microarray analysis of genes expressed in ripened receptacle did not show any significant change between control and transgenic fruits. Thus, contrary to the most accepted view, it is concluded that PG plays a key role on pectin metabolism and softening of strawberry fruit. PMID:19820312

The polygalacturonase FaPG1 gene plays a key role in strawberry fruit softening.

PubMed

García-Gago, Juan A; Posé, Sara; Muñoz-Blanco, Juan; Quesada, Miguel A; Mercado, José A

2009-08-01

The loss of firm texture is one of the most characteristic physiological processes that occur during the ripening of fleshy fruits. It is generally accepted that the disassembly of primary cell wall and middle lamella is the main factor involved in fruit softening. In this process, polygalacturonase (PG) has been implicated in the degradation of the polyuronide network in several fruits. However, the minor effect of PG downregulation on tomato softening, reported during the nineties, minimized the role of this enzyme in softening. Further works in other fruits are challenging this general assumption, as is occurring in strawberry. The strawberry (Fragaria x ananassa) fruit undergoes an extensive and fast softening that limit its shelf life and postharvest. Traditionally, it has also been considered that PG plays a minor role on this process, due to the low PG activity found in ripened strawberry fruits. Transgenic strawberry plants expressing an antisense sequence of the ripening-specific PG gene FaPG1 have been generated to get an insight into the role of this gene in softening. Half of the transgenic lines analyzed yielded fruits significantly firmer than control, without being affected other fruit parameters such as weight, color or soluble solids. The increase on firmness was maintained after several days of posharvest. In these firmer lines, FaPG1 was silenced to 95%, but total PG activity was only minor reduced. At the cell wall level, transgenic fruits contained a higher amount of covalently bound pectins whereas the soluble fraction was diminished. A microarray analysis of genes expressed in ripened receptacle did not show any significant change between control and transgenic fruits. Thus, contrary to the most accepted view, it is concluded that PG plays a key role on pectin metabolism and softening of strawberry fruit.

Surface characterization of colloidal-sol gel derived biphasic HA/FA coatings.

PubMed

Cheng, Kui; Zhang, Sam; Weng, Wenjian

2007-10-01

Hydroxyapatite (HA) powders are ultrasonically dispersed in the precursor of fluoridated hydroxyapatite (FHA) or fluorapatite (FA) to form a "colloidal sol". HA/FA biphasic coatings are prepared on Ti6Al4V substrate via dip coating, 150 degrees C drying and 600 degrees C firing. The coatings show homogenous distribution of HA particles in the FA matrix. The relative phase proportion can be tailored by the amount of HA in the colloidal sol. The surfaces of the coatings consist of two kinds of distinct domains: HA and FA, resulting in a compositionally heterogeneous surface. The biphasic coating surface becomes increasingly rougher with HA powders, from around 200 nm of pure FA to 400-600 nm in Ra of biphasic coatings. The rougher biphasic HA/FA surfaces with chemically controllable domains will favor cell attachment, apatite layer deposition and necessary dissolution in clinical applications.

FaPOD27 functions in the metabolism of polyphenols in strawberry fruit (Fragaria sp.)

PubMed Central

Yeh, Su-Ying; Huang, Fong-Chin; Hoffmann, Thomas; Mayershofer, Mechthild; Schwab, Wilfried

2014-01-01

The strawberry (Fragaria × ananassa) is one of the most preferred fresh fruit worldwide, accumulates numerous flavonoids but has limited shelf life due to excessive tissue softening caused by cell wall degradation. Since lignin is one of the polymers that strengthen plant cell walls and might contribute to some extent to fruit firmness monolignol biosynthesis was studied in strawberry fruit. Cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD), and a peroxidase (POD27) gene were strongly expressed in red, ripe fruit whereas a second POD gene was primarily expressed in green, immature fruit. Moreover, FaPOD27 transcripts were strongly and constitutively induced in fruits exposed to Agrobacterium infection. Gene expression levels and enzymatic activities of FaCCR and FaCAD were efficiently suppressed through RNAi in FaCCR- and FaCAD-silenced strawberries. Besides, significantly elevated FaPOD transcript levels were detected after agroinfiltration of pBI-FaPOD constructs in fruits. At the same time, levels of G-monomers were considerably reduced in FaCCR-silenced fruits whereas the proportion of both G- and S-monomers decisively decreased in FaCAD-silenced and pBI-FaPOD fruits. Development, firmness, and lignin level of the treated fruits were similar to pBI-intron control fruits, presumably attributed to increased expression levels of FaPOD27 upon agroinfiltration. Additionally, enhanced firmness, accompanied with elevated lignin levels, was revealed in chalcone synthase-deficient fruits (CHS−), independent of down- or up-regulation of individual and combined FaCCR. FaCAD, and FaPOD genes by agroinfiltration, when compared to CHS−/pBI-intron control fruits. These approaches provide further insight into the genetic control of flavonoid and lignin synthesis in strawberries. The results suggest that FaPOD27 is a key gene for lignin biosynthesis in strawberry fruit and thus to improving the firmness of strawberries. PMID:25346738

Genome Sequence of the Alkaline-Tolerant Cellulomonas sp. Strain FA1

DOE PAGES

Cohen, Michael F.; Hu, Ping; Nguyen, My Vu; ...

2015-06-18

We present the genome of the cellulose-degrading Cellulomonas sp. strain FA1 isolated from an actively serpentinizing highly alkaline spring. Knowledge of this genome will enable studies into the molecular basis of plant material degradation in alkaline environments and inform the development of lignocellulose bioprocessing procedures for biofuel production.

Overexpression of a BAHD Acyltransferase, OsAt10, Alters Rice Cell Wall Hydroxycinnamic Acid Content and Saccharification1[C][W][OA

PubMed Central

Bartley, Laura E.; Peck, Matthew L.; Kim, Sung-Ryul; Ebert, Berit; Manisseri, Chithra; Chiniquy, Dawn M.; Sykes, Robert; Gao, Lingfang; Rautengarten, Carsten; Vega-Sánchez, Miguel E.; Benke, Peter I.; Canlas, Patrick E.; Cao, Peijian; Brewer, Susan; Lin, Fan; Smith, Whitney L.; Zhang, Xiaohan; Keasling, Jay D.; Jentoff, Rolf E.; Foster, Steven B.; Zhou, Jizhong; Ziebell, Angela; An, Gynheung; Scheller, Henrik V.; Ronald, Pamela C.

2013-01-01

Grass cell wall properties influence food, feed, and biofuel feedstock usage efficiency. The glucuronoarabinoxylan of grass cell walls is esterified with the phenylpropanoid-derived hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA). Feruloyl esters undergo oxidative coupling with neighboring phenylpropanoids on glucuronoarabinoxylan and lignin. Examination of rice (Oryza sativa) mutants in a grass-expanded and -diverged clade of BAHD acyl-coenzyme A-utilizing transferases identified four mutants with altered cell wall FA or p-CA contents. Here, we report on the effects of overexpressing one of these genes, OsAt10 (LOC_Os06g39390), in rice. An activation-tagged line, OsAT10-D1, shows a 60% reduction in matrix polysaccharide-bound FA and an approximately 300% increase in p-CA in young leaf tissue but no discernible phenotypic alterations in vegetative development, lignin content, or lignin composition. Two additional independent OsAt10 overexpression lines show similar changes in FA and p-CA content. Cell wall fractionation and liquid chromatography-mass spectrometry experiments isolate the cell wall alterations in the mutant to ester conjugates of a five-carbon sugar with p-CA and FA. These results suggest that OsAT10 is a p-coumaroyl coenzyme A transferase involved in glucuronoarabinoxylan modification. Biomass from OsAT10-D1 exhibits a 20% to 40% increase in saccharification yield depending on the assay. Thus, OsAt10 is an attractive target for improving grass cell wall quality for fuel and animal feed. PMID:23391577

Differential Protection of Cry1Fa Toxin against Spodoptera frugiperda Larval Gut Proteases by Cadherin Orthologs Correlates with Increased Synergism

PubMed Central

Rahman, Khalidur; Abdullah, Mohd Amir F.; Ambati, Suresh; Taylor, Milton D.

2012-01-01

The Cry proteins produced by Bacillus thuringiensis (Bt) are the most widely used biopesticides effective against a range of crop pests and disease vectors. Like chemical pesticides, development of resistance is the primary threat to the long-term efficacy of Bt toxins. Recently discovered cadherin-based Bt Cry synergists showed the potential to augment resistance management by improving efficacy of Cry toxins. However, the mode of action of Bt Cry synergists is thus far unclear. Here we elucidate the mechanism of cadherin-based Cry toxin synergism utilizing two cadherin peptides, Spodoptera frugiperda Cad (SfCad) and Manduca sexta Cad (MsCad), which differentially enhance Cry1Fa toxicity to Spodoptera frugiperda neonates. We show that differential SfCad- and MsCad-mediated protection of Cry1Fa toxin in the Spodoptera frugiperda midgut correlates with differential Cry1Fa toxicity enhancement. Both peptides exhibited high affinity for Cry1Fa toxin and an increased rate of Cry1Fa-induced pore formation in S. frugiperda. However, only SfCad bound the S. frugiperda brush border membrane vesicle and more effectively prolonged the stability of Cry1Fa toxin in the gut, explaining higher Cry1Fa enhancement by this peptide. This study shows that cadherin fragments may enhance B. thuringiensis toxicity by at least two different mechanisms or a combination thereof: (i) protection of Cry toxin from protease degradation in the insect midgut and (ii) enhancement of pore-forming ability of Cry toxin. PMID:22081566

Differential protection of Cry1Fa toxin against Spodoptera frugiperda larval gut proteases by cadherin orthologs correlates with increased synergism.

PubMed

Rahman, Khalidur; Abdullah, Mohd Amir F; Ambati, Suresh; Taylor, Milton D; Adang, Michael J

2012-01-01

The Cry proteins produced by Bacillus thuringiensis (Bt) are the most widely used biopesticides effective against a range of crop pests and disease vectors. Like chemical pesticides, development of resistance is the primary threat to the long-term efficacy of Bt toxins. Recently discovered cadherin-based Bt Cry synergists showed the potential to augment resistance management by improving efficacy of Cry toxins. However, the mode of action of Bt Cry synergists is thus far unclear. Here we elucidate the mechanism of cadherin-based Cry toxin synergism utilizing two cadherin peptides, Spodoptera frugiperda Cad (SfCad) and Manduca sexta Cad (MsCad), which differentially enhance Cry1Fa toxicity to Spodoptera frugiperda neonates. We show that differential SfCad- and MsCad-mediated protection of Cry1Fa toxin in the Spodoptera frugiperda midgut correlates with differential Cry1Fa toxicity enhancement. Both peptides exhibited high affinity for Cry1Fa toxin and an increased rate of Cry1Fa-induced pore formation in S. frugiperda. However, only SfCad bound the S. frugiperda brush border membrane vesicle and more effectively prolonged the stability of Cry1Fa toxin in the gut, explaining higher Cry1Fa enhancement by this peptide. This study shows that cadherin fragments may enhance B. thuringiensis toxicity by at least two different mechanisms or a combination thereof: (i) protection of Cry toxin from protease degradation in the insect midgut and (ii) enhancement of pore-forming ability of Cry toxin.

Shared Midgut Binding Sites for Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac and Cry1Fa Proteins from Bacillus thuringiensis in Two Important Corn Pests, Ostrinia nubilalis and Spodoptera frugiperda

PubMed Central

Hernández-Rodríguez, Carmen Sara; Hernández-Martínez, Patricia; Van Rie, Jeroen; Escriche, Baltasar; Ferré, Juan

2013-01-01

First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV) from last instar larval midguts were used in competition binding assays with 125I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case. PMID:23861865

Fanconi anemia mesenchymal stromal cells-derived glycerophospholipids skew hematopoietic stem cell differentiation through Toll-like receptor signaling

PubMed Central

Amarachintha, Surya; Sertorio, Mathieu; Wilson, Andrew; Li, Xiaoli; Pang, Qishen

2015-01-01

Fanconi anemia (FA) patients develop bone marrow (BM) failure or leukemia. One standard care for these devastating complications is hematopoietic stem cell transplantation. We identified a group of mesenchymal stromal cells (MSCs)-derived metabolites, glycerophospholipids and their endogenous inhibitor, 5-(Tetradecyloxy)-2-furoic acid (TOFA), as regulators of donor hematopoietic stem and progenitor cells (HSPCs). We provided two pieces of evidence that TOFA could improve hematopoiesis-supporting function of FA MSCs: (1) limiting-dilution CAFC assay revealed that TOFA significantly increased cobblestone colonies in Fanca−/− or Fancd2−/− co-cultures compared to untreated co-cultures. (2) Competitive repopulating assay using output cells collected from co-cultures showed that TOFA greatly alleviated the abnormal expansion of the donor myeloid (CD45.2+Gr1+Mac1+) compartment in both peripheral blood and BM of recipient mice transplanted with cells from Fanca−/− or Fancd2−/− co-cultures. Further, mechanistic studies identified Tlr4 signaling as the responsible pathway mediating the effect of glycerophospholipids. Thus, targeting Glycerophospholipid biosynthesis in FA MSCs could be a therapeutic strategy to improve hematopoiesis and stem cell transplantation. PMID:26212365

CADM1/TSLC1 Identifies HTLV-1-Infected Cells and Determines Their Susceptibility to CTL-Mediated Lysis

PubMed Central

Tanaka, Yuetsu; Taylor, Graham P.; Bangham, Charles R. M.

2016-01-01

Human T cell lymphotropic virus-1 (HTLV-1) primarily infects CD4+ T cells, causing inflammatory disorders or a T cell malignancy in 5% to 10% of carriers. The cytotoxic T lymphocyte (CTL) response is a key factor that controls the viral load and thus the risk of disease. The ability to detect the viral protein Tax in primary cells has made it possible to estimate the rate at which Tax-expressing infected cells are eliminated by CTLs in persistently infected people. However, most HTLV-1-infected cells are Tax–at a given time, and their immunophenotype is poorly defined. Here, we aimed to identify a cell-surface molecule expressed by both Tax+ and Tax–HTLV-1-infected cells and use it to analyse the CTL response in fresh peripheral blood mononuclear cells. Cell adhesion molecule 1 (CADM1/TSLC1) was the best single marker of HTLV-1 infection, identifying HTLV-1-infected cells with greater sensitivity and specificity than CD25, CCR4 or ICAM-1. CADM1+CD4+ T cells carried a median of 65% of proviral copies in peripheral blood. In a cohort of 23 individuals, we quantified the rate of CTL-mediated killing of Tax+ and Tax−CADM1+ cells. We show that CADM1 expression is associated with enhanced susceptibility of infected cells to CTL lysis: despite the immunodominance of Tax in the CTL response, Tax+CADM1– cells were inefficiently lysed by CTLs. Upregulation of the CADM1 ligand CRTAM on CD8+ T cells correlated with efficient lysis of infected cells. Tax–CADM1+ cells were lysed at a very low rate by autologous CTLs, however, were efficiently killed when loaded with exogenous peptide antigen. High expression of CADM1 on most HTLV-1-infected cells in the face of enhanced CTL counterselection implies that CADM1 confers a strong benefit on the virus. PMID:27105228

A never-ending story: the steadily growing family of the FA and FA-like genes

PubMed Central

Gueiderikh, Anna; Rosselli, Filippo; Neto, Januario B.C.

2017-01-01

Abstract Among the chromosome fragility-associated human syndromes that present cancer predisposition, Fanconi anemia (FA) is unique due to its large genetic heterogeneity. To date, mutations in 21 genes have been associated with an FA or an FA-like clinical and cellular phenotype, whose hallmarks are bone marrow failure, predisposition to acute myeloid leukemia and a cellular and chromosomal hypersensitivity to DNA crosslinking agents exposure. The goal of this review is to trace the history of the identification of FA genes, a history that started in the eighties and is not yet over, as indicated by the cloning of a twenty-first FA gene in 2016. PMID:28558075

Induction of plasminogen activator inhibitor type-1 (PAI-1) by hypoxia and irradiation in human head and neck carcinoma cell lines

PubMed Central

Schilling, Daniela; Bayer, Christine; Geurts-Moespot, Anneke; Sweep, Fred CGJ; Pruschy, Martin; Mengele, Karin; Sprague, Lisa D; Molls, Michael

2007-01-01

Background Squamous cell carcinoma of the head and neck (SCCHN) often contain highly radioresistant hypoxic regions, nonetheless, radiotherapy is a common treatment modality for these tumours. Reoxygenation during fractionated radiotherapy is desired to render these hypoxic tumour regions more radiosensitive. Hypoxia additionally leads to up-regulation of PAI-1, a protein involved in tumour progression and an established prognostic marker for poor outcome. However, the impact of reoxygenation and radiation on PAI-1 levels is not yet clear. Therefore, we investigated the kinetics of PAI-1 expression and secretion after hypoxia and reoxygenation, and determined the influence of ionizing radiation on PAI-1 levels in the two human SCCHN cell lines, BHY and FaDu. Methods HIF-1α immunoblot was used to visualize the degree of hypoxia in the two cell lines. Cellular PAI-1 expression was investigated by immunofluorescence microscopy. ELISA was used to quantify relative changes in PAI-1 expression (cell lysates) and secretion (cell culture supernatants) in response to various lengths (2 – 4 h) of hypoxic exposure (< 0.66 % O2), reoxygenation (24 h, 20 % O2), and radiation (0, 2, 5 and 10 Gy). Results HIF-1α expression was induced between 2 and 24 h of hypoxic exposure. Intracellular PAI-1 expression was significantly increased in BHY and FaDu cells as early as 4 h after hypoxic exposure. A significant induction in secreted PAI-1 was seen after 12 to 24 h (BHY) and 8 to 24 h (FaDu) hypoxia, as compared to the normoxic control. A 24 h reoxygenation period caused significantly less PAI-1 secretion than a 24 h hypoxia period in FaDu cells. Irradiation led to an up-regulation of PAI-1 expression and secretion in both, BHY and FaDu cells. Conclusion Our data suggest that both, short-term (~4 – 8 h) and long-term (~20 – 24 h) hypoxic exposure could increase PAI-1 levels in SCCHN in vivo. Importantly, radiation itself could lead to PAI-1 up-regulation in head and neck

Induction of plasminogen activator inhibitor type-1 (PAI-1) by hypoxia and irradiation in human head and neck carcinoma cell lines.

PubMed

Schilling, Daniela; Bayer, Christine; Geurts-Moespot, Anneke; Sweep, Fred C G J; Pruschy, Martin; Mengele, Karin; Sprague, Lisa D; Molls, Michael

2007-07-30

Squamous cell carcinoma of the head and neck (SCCHN) often contain highly radioresistant hypoxic regions, nonetheless, radiotherapy is a common treatment modality for these tumours. Reoxygenation during fractionated radiotherapy is desired to render these hypoxic tumour regions more radiosensitive. Hypoxia additionally leads to up-regulation of PAI-1, a protein involved in tumour progression and an established prognostic marker for poor outcome. However, the impact of reoxygenation and radiation on PAI-1 levels is not yet clear. Therefore, we investigated the kinetics of PAI-1 expression and secretion after hypoxia and reoxygenation, and determined the influence of ionizing radiation on PAI-1 levels in the two human SCCHN cell lines, BHY and FaDu. HIF-1alpha immunoblot was used to visualize the degree of hypoxia in the two cell lines. Cellular PAI-1 expression was investigated by immunofluorescence microscopy. ELISA was used to quantify relative changes in PAI-1 expression (cell lysates) and secretion (cell culture supernatants) in response to various lengths (2-4 h) of hypoxic exposure (< 0.66% O2), reoxygenation (24 h, 20% O2), and radiation (0, 2, 5 and 10 Gy). HIF-1alpha expression was induced between 2 and 24 h of hypoxic exposure. Intracellular PAI-1 expression was significantly increased in BHY and FaDu cells as early as 4 h after hypoxic exposure. A significant induction in secreted PAI-1 was seen after 12 to 24 h (BHY) and 8 to 24 h (FaDu) hypoxia, as compared to the normoxic control. A 24 h reoxygenation period caused significantly less PAI-1 secretion than a 24 h hypoxia period in FaDu cells. Irradiation led to an up-regulation of PAI-1 expression and secretion in both, BHY and FaDu cells. Our data suggest that both, short-term (approximately 4-8 h) and long-term (approximately 20-24 h) hypoxic exposure could increase PAI-1 levels in SCCHN in vivo. Importantly, radiation itself could lead to PAI-1 up-regulation in head and neck tumours, whereas

Alterations of local cerebral glucose utilization in lean and obese fa/fa rats after acute adrenalectomy.

PubMed

Doyle, P; Rohner-Jeanrenaud, F; Jeanrenaud, B

1994-08-29

An animal model often used to investigate the aetiology of obesity is the genetically obese fa/fa rat. It has many abnormalities, including hyperphagia, hyper-insulinemia, insulin resistance, low cerebral glucose utilization and an overactive hypothalamo-pituitary adrenal (HPA) axis with resulting hypercorticism. Due to the latter consideration, the aim of this work was to study the impact of acute adrenalectomy (ADX) on the local cerebral glucose utilization (LCGU) of lean and obese fa/fa rats. ADX resulted in discrete increases in LCGU of regions common to both lean and obese rats. These common regions were found to belong to be related to the limbic system. Within this system, the LCGU of the brain of obese rats was either normalized to lean sham operated values or increased by ADX to a similar degree in both groups on a percentage basis. It was concluded that the LCGU of both lean and obese animals appears to be negatively regulated, albeit to different extents, by glucocorticoids. Such negative regulation is particularly salient within the limbic system of the lean rat and even more so in the fa/fa rat. It is suggested that the long-term hypercorticism of obese fa/fa rats due to abnormal regulation of the HPA axis may result in a decreased LCGU in limbic and related regions of the brain of fa/fa rats and contribute to the expression of the obese phenotype.

Global emissions of the hydrofluorocarbons (HFCs) HFC-365mfc, HFC-245fa, HFC-227ea, and HFC-236fa based on atmospheric observations

NASA Astrophysics Data System (ADS)

Vollmer, M. K.; Miller, B. R.; Rigby, M. L.; Reimann, S.; Muhle, J.; Agage, Soge, Snu Members, Kopri Members

2010-12-01

We report on the atmospheric measurements and global emissions of the hydrofluorocarbons (HFCs) HFC-365mfc (CH3CH2CF2CF3, 1,1,1,3,3-pentafluorobutane), HFC-245fa (CHF2CH2CF3, 1,1,1,3,3-pentafluoropropane), HFC-227ea (CF3CHFCF3, 1,1,1,2,3,3,3-heptafluoropropane), and HFC-236fa (CF3CH2CF3, 1,1,1,3,3,3-hexafluoropropane). These measurements are from in-situ observations at stations of AGAGE (Advanced Global Atmospheric Gases Experiment) and SOGE (System for Observations of Halogenated Greenhouse Gases in Europe), and from the Korean station Gosan. We also report on flask sample measurements from the Antarctic stations King Sejong and Troll, and extend our records back to the 1970s using archived air samples of both hemispheres. All data are used in a global 12-box 2-dimensional atmospheric transport model to derive global abundances and emission estimates. All four HFCs have strongly increased in the atmosphere in recent years with growth rates at nearly 10 %, resulting in dry air mole fractions at the end of 2009 of 0.49 ppt for HFC-365mfc, 1.00 ppt for HFC-245fa, and 0.51 ppt for HFC-227ea. HFC-236fa, for which we report the first atmospheric measurements, is less abundant and has grown to 0.069 ppt at the end of 2009. Our model results show rapidly growing emissions of HFC-365mfc and HFC-245fa after 2002 but surprisingly these have now started to decline to globally 2.7 kt/yr (HFC-365mfc) and 6.1 kt/yr (HFC-245fa). On the other hand HFC-227ea and HFC-236fa show uninterrupted growth in their emissions of 2.5 kt/yr and 0.2 kt/yr at the end of 2009.

Δ(9)-THC modulation of fatty acid 2-hydroxylase (FA2H) gene expression: possible involvement of induced levels of PPARα in MDA-MB-231 breast cancer cells.

PubMed

Takeda, Shuso; Ikeda, Eriko; Su, Shengzhong; Harada, Mari; Okazaki, Hiroyuki; Yoshioka, Yasushi; Nishimura, Hajime; Ishii, Hiroyuki; Kakizoe, Kazuhiro; Taniguchi, Aya; Tokuyasu, Miki; Himeno, Taichi; Watanabe, Kazuhito; Omiecinski, Curtis J; Aramaki, Hironori

2014-12-04

We recently reported that Δ(9)-tetrahydrocannabinol (Δ(9)-THC), a major cannabinoid component in Cannabis Sativa (marijuana), significantly stimulated the expression of fatty acid 2-hydroxylase (FA2H) in human breast cancer MDA-MB-231 cells. Peroxisome proliferator-activated receptor α (PPARα) was previously implicated in this induction. However, the mechanisms mediating this induction have not been elucidated in detail. We performed a DNA microarray analysis of Δ(9)-THC-treated samples and showed the selective up-regulation of the PPARα isoform coupled with the induction of FA2H over the other isoforms (β and γ). Δ(9)-THC itself had no binding/activation potential to/on PPARα, and palmitic acid (PA), a PPARα ligand, exhibited no stimulatory effects on FA2H in MDA-MB-231 cells; thus, we hypothesized that the levels of PPARα induced were involved in the Δ(9)-THC-mediated increase in FA2H. In support of this hypothesis, we herein demonstrated that; (i) Δ(9)-THC activated the basal transcriptional activity of PPARα in a concentration-dependent manner, (ii) the concomitant up-regulation of PPARα/FA2H was caused by Δ(9)-THC, (iii) PA could activate PPARα after the PPARα expression plasmid was introduced, and (iv) the Δ(9)-THC-induced up-regulation of FA2H was further stimulated by the co-treatment with L-663,536 (a known PPARα inducer). Taken together, these results support the concept that the induced levels of PPARα may be involved in the Δ(9)-THC up-regulation of FA2H in MDA-MB-231 cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Dysfunction of protein kinase FA/GSK-3 alpha in lymphocytes of patients with schizophrenic disorder.

PubMed

Yang, S D; Yu, J S; Lee, T T; Yang, C C; Ni, M H; Yang, Y Y

1995-09-01

As compared to normal people, the lymphocytes of patients with schizophrenia were found to have an impairment of ATP.Mg-dependent protein phosphatase activation. More importantly, the impaired protein phosphatase activation in the lymphocytes of schizophrenic patients could be consistently and completely restored to normal by exogenous pure protein kinase FA/glycogen synthase kinase-3 alpha (kinase FA/GSK-3 alpha) (the activating factor of ATP.Mg-dependent protein phosphatase), indicating that the molecular mechanism for the impaired protein phosphatase activation in schizophrenic patients may be due to a functional loss of kinase FA/GSK-3 alpha. Immunoblotting and kinase activity analysis in an anti-kinase FA/GSK-3 alpha immunoprecipitate further demonstrate that both cellular activities and protein levels of kinase FA/GSK-3 alpha in the lymphocytes of schizophrenic patients were greatly impared as compared to normal controls. Statistical analysis revealed that the lymphocytes isolated from 37 normal people contain kinase FA/GSK-3 alpha activity in the high levels of 14.8 +/- 2.4 units/mg of cell protein, whereas the lymphocytes of 48 patients with schizophrenic disorder contain kinase FA/GSK-3 alpha activity in the low levels of 2.8 +/- 1.6 units/mg, indicating that the different levels of kinase FA/GSK-3 alpha activity between schizophrenic patients and normal people are statistically significant. Taken together, the results provide initial evidence that patients with schizophrenic disorder may have a common impairment in the protein levels and cellular activities of kinase FA/GSK-3 alpha, a multisubstrate protein kinase and a multisubstrate protein phosphatase activator in their lymphocytes.

Regulation of palmitoyl-CoA chain elongation by clofibric acid in the liver of Zucker fa/fa rats.

PubMed

Toyama, Tomoaki; Kudo, Naomi; Mitsumoto, Atsushi; Kawashima, Yoichi

2005-05-01

The regulation of palmitoyl-CoA chain elongation (PCE) by clofibric acid [2-(4-chlorophenoxy)-2-methylpropionic acid] was investigated in comparison with stearoyl-CoA desaturase (SCD) in the liver of obese Zucker fa/fa rats. The proportion of oleic acid in the hepatic lipids of Zucker obese rats is 2.7 times higher than that of lean littermates. The activities of PCE and SCD in the liver of Zucker obese rats were markedly higher than in lean rats, and the hepatic uptake of 2-deoxyglucose (2-DG) was also higher in Zucker obese rats compared with lean rats. The increased activities of SCD and PCE in Zucker obese rats were due to the enhanced expression of mRNA of both SCD1 and rat FA elongase 2 (rELO2), but not SCD2 or rELO1. The proportion of oleic acid in the liver was significantly increased by the administration of clofibric acid to Zucker obese rats, and the hepatic PCE activity and rELO2 mRNA expression, but not the SCD activity or SCD1 mRNA expression, were increased in response to clofibric acid treatment. By contrast, the activities of both PCE and SCD and the mRNA expression of SCD1 and rELO2 in the liver were increased by the treatment of Zucker lean rats with clofibric acid. Multiple regression analysis, which was performed to determine the relationships involving PCE activity, SCD activity, and the proportion of oleic acid, revealed that the three parameters were significantly correlated and that the standardized partial regression coefficient of PCE was higher than that of SCD. These results indicate that oleic acid is synthesized by the concerted action of PCE and SCD and that PCE plays a crucial role in the formation of oleic acid when Zucker fa/fa rats are given clofibric acid.

Atmospheric histories and global emissions of the anthropogenic hydrofluorocarbons HFC-365mfc, HFC-245fa, HFC-227ea, and HFC-236fa

NASA Astrophysics Data System (ADS)

Vollmer, Martin K.; Miller, Benjamin R.; Rigby, Matthew; Reimann, Stefan; Mühle, Jens; Krummel, Paul B.; O'Doherty, Simon; Kim, Jooil; Rhee, Tae Siek; Weiss, Ray F.; Fraser, Paul J.; Simmonds, Peter G.; Salameh, Peter K.; Harth, Christina M.; Wang, Ray H. J.; Steele, L. Paul; Young, Dickon; Lunder, Chris R.; Hermansen, Ove; Ivy, Diane; Arnold, Tim; Schmidbauer, Norbert; Kim, Kyung-Ryul; Greally, Brian R.; Hill, Matthias; Leist, Michael; Wenger, Angelina; Prinn, Ronald G.

2011-04-01

We report on ground-based atmospheric measurements and emission estimates of the four anthropogenic hydrofluorocarbons (HFCs) HFC-365mfc (CH3CF2CH2CF3, 1,1,1,3,3-pentafluorobutane), HFC-245fa (CHF2CH2CF3, 1,1,1,3,3-pentafluoropropane), HFC-227ea (CF3CHFCF3, 1,1,1,2,3,3,3-heptafluoropropane), and HFC-236fa (CF3CH2CF3, 1,1,1,3,3,3-hexafluoropropane). In situ measurements are from the global monitoring sites of the Advanced Global Atmospheric Gases Experiment (AGAGE), the System for Observations of Halogenated Greenhouse Gases in Europe (SOGE), and Gosan (South Korea). We include the first halocarbon flask sample measurements from the Antarctic research stations King Sejong and Troll. We also present measurements of archived air samples from both hemispheres back to the 1970s. We use a two-dimensional atmospheric transport model to simulate global atmospheric abundances and to estimate global emissions. HFC-365mfc and HFC-245fa first appeared in the atmosphere only ˜1 decade ago; they have grown rapidly to globally averaged dry air mole fractions of 0.53 ppt (in parts per trillion, 10-12) and 1.1 ppt, respectively, by the end of 2010. In contrast, HFC-227ea first appeared in the global atmosphere in the 1980s and has since grown to ˜0.58 ppt. We report the first measurements of HFC-236fa in the atmosphere. This long-lived compound was present in the atmosphere at only 0.074 ppt in 2010. All four substances exhibit yearly growth rates of >8% yr-1 at the end of 2010. We find rapidly increasing emissions for the foam-blowing compounds HFC-365mfc and HFC-245fa starting in ˜2002. After peaking in 2006 (HFC-365mfc: 3.2 kt yr-1, HFC-245fa: 6.5 kt yr-1), emissions began to decline. Our results for these two compounds suggest that recent estimates from long-term projections (to the late 21st century) have strongly overestimated emissions for the early years of the projections (˜2005-2010). Global HFC-227ea and HFC-236fa emissions have grown to average values of 2.4 kt yr-1

A Novel Hydroxamate-Based Compound WMJ-J-09 Causes Head and Neck Squamous Cell Carcinoma Cell Death via LKB1-AMPK-p38MAPK-p63-Survivin Cascade.

PubMed

Yen, Chia-Sheng; Choy, Cheuk-Sing; Huang, Wei-Jan; Huang, Shiu-Wen; Lai, Pin-Ye; Yu, Meng-Chieh; Shiue, Ching; Hsu, Ya-Fen; Hsu, Ming-Jen

2018-01-01

Growing evidence shows that hydroxamate-based compounds exhibit broad-spectrum pharmacological properties including anti-tumor activity. However, the precise mechanisms underlying hydroxamate derivative-induced cancer cell death remain incomplete understood. In this study, we explored the anti-tumor mechanisms of a novel aliphatic hydroxamate-based compound, WMJ-J-09, in FaDu head and neck squamous cell carcinoma (HNSCC) cells. WMJ-J-09 induced G2/M cell cycle arrest and apoptosis in FaDu cells. These actions were associated with liver kinase B1 (LKB1), AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (p38MAPK) activation, transcription factor p63 phosphorylation, as well as modulation of p21 and survivin. LKB1-AMPK-p38MAPK signaling blockade reduced WMJ-J-09's enhancing effects in p63 phosphorylation, p21 elevation and survivin reduction. Moreover, WMJ-J-09 caused an increase in α-tubulin acetylation and interfered with microtubule assembly. Furthermore, WMJ-J-09 suppressed the growth of subcutaneous FaDu xenografts in vivo . Taken together, WMJ-J-09-induced FaDu cell death may involve LKB1-AMPK-p38MAPK-p63-survivin signaling cascade. HDACs inhibition and disruption of microtubule assembly may also contribute to WMJ-J-09's actions in FaDu cells. This study suggests that WMJ-J-09 may be a potential lead compound and warrant the clinical development in the treatment of HNSCC.

Ferulic acid suppresses activation of hepatic stellate cells through ERK1/2 and Smad signaling pathways in vitro.

PubMed

Xu, Tianjiao; Pan, Zhi; Dong, Miaoxian; Yu, Chunlei; Niu, Yingcai

2015-01-01

Hepatic stellate cells (HSCs) are the primary source of matrix components in hepatic fibrosis. Ferulic acid (FA) has antifibrotic potential in renal and cardiac disease. However, whether FA comprises inhibitive effects of HSCs activation remains to be clarified. This study aims at evaluating the hypothesis that FA inhibits extracellular matrix (ECM)-related gene expression by the interruption of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) or/and Smad signaling pathways in HSC-T6. Our results indicated that FA significantly inhibited both viability and activation of HSC-T6 cells in vitro. In addition, we demonstrated, for the first time, that FA dramatically inhibited the expression of α1(I) collagen (Col-I) and fibronectin at levels of transcription and translation. Moreover, FA treatment inhibited Smad transcriptional activity, as evaluated by transient transfection with a plasmid construction containing SMAD response element and the luciferase reporter gene. Furthermore, FA inhibition of HSCs activation involved in both focal adhesion kinase (FAK)-dependent ERK1/2 and Smad signaling pathways with independent manner. Blocking transforming growth factor-β by a neutralizing antibody caused a marked reduction in both ERK1/2 and Smad signaling. These results support FA as an effective therapeutic agent for the prevention and treatment of hepatic fibrosis. Copyright © 2014 Elsevier Inc. All rights reserved.

Hydrogen Sulfide Inhibits Formaldehyde-Induced Endoplasmic Reticulum Stress in PC12 Cells by Upregulation of SIRT-1

PubMed Central

Zhang, Ping; Chen, Li-Xun; Wang, Li; Xie, Ming; Wang, Chun-Yan; Tang, Xiao-Qing

2014-01-01

Background Formaldehyde (FA), a well-known environmental pollutant, has been classified as a neurotoxic molecule. Our recent data demonstrate that hydrogen sulfide (H2S), the third gaseous transmitter, has a protective effect on the neurotoxicity of FA. However, the exact mechanisms underlying this protection remain largely unknown. Endoplasmic reticulum (ER) stress has been implicated in the neurotoxicity of FA. Silent mating type information regulator 2 homolog 1 (SIRT-1), a histone deacetylases, has various biological activities, including the extension of lifespan, the modulation of ER stress, and the neuroprotective action. Objective We hypothesize that the protection of H2S against FA-induced neurotoxicity involves in inhibiting ER stress by upregulation of SIRT-1. The present study attempted to investigate the protective effect of H2S on FA-induced ER stress in PC12 cells and the contribution of SIRT-1 to the protection of H2S against FA-induced injuries, including ER stress, cytotoxicity and apoptosis. Principal Findings We found that exogenous application of sodium hydrosulfide (NaHS; an H2S donor) significantly attenuated FA-induced ER stress responses, including the upregulated levels of glucose-regulated protein 78, C/EBP homologous protein, and cleaved caspase-12 expression. We showed that NaHS upregulates the expression of SIRT-1 in PC12 cells. Moreover, the protective effects of H2S on FA-elicited ER stress, cytotoxicity and apoptosis were reversed by Sirtinol, a specific inhibitor of SIRT-1. Conclusion/Significance These data indicate that H2S exerts its protection against the neurotoxicity of FA through overcoming ER stress via upregulation of SIRT-1. Our findings provide novel insights into the protective mechanisms of H2S against FA-induced neurotoxicity. PMID:24587076

Cell surface marker profiling of human tracheal basal cells reveals distinct subpopulations, identifies MST1/MSP as a mitogenic signal, and identifies new biomarkers for lung squamous cell carcinomas.

PubMed

Van de Laar, Emily; Clifford, Monica; Hasenoeder, Stefan; Kim, Bo Ram; Wang, Dennis; Lee, Sharon; Paterson, Josh; Vu, Nancy M; Waddell, Thomas K; Keshavjee, Shaf; Tsao, Ming-Sound; Ailles, Laurie; Moghal, Nadeem

2014-12-31

The large airways of the lungs (trachea and bronchi) are lined with a pseudostratified mucociliary epithelium, which is maintained by stem cells/progenitors within the basal cell compartment. Alterations in basal cell behavior can contribute to large airway diseases including squamous cell carcinomas (SQCCs). Basal cells have traditionally been thought of as a uniform population defined by basolateral position, cuboidal cell shape, and expression of pan-basal cell lineage markers like KRT5 and TP63. While some evidence suggests that basal cells are not all functionally equivalent, few heterogeneously expressed markers have been identified to purify and study subpopulations. In addition, few signaling pathways have been identified that regulate their cell behavior. The goals of this work were to investigate tracheal basal cell diversity and to identify new signaling pathways that regulate basal cell behavior. We used flow cytometry (FACS) to profile cell surface marker expression at a single cell level in primary human tracheal basal cell cultures that maintain stem cell/progenitor activity. FACS results were validated with tissue staining, in silico comparisons with normal basal cell and lung cancer datasets, and an in vitro proliferation assay. We identified 105 surface markers, with 47 markers identifying potential subpopulations. These subpopulations generally fell into more (~ > 13%) or less abundant (~ < 6%) groups. Microarray gene expression profiling supported the heterogeneous expression of these markers in the total population, and immunostaining of large airway tissue suggested that some of these markers are relevant in vivo. 24 markers were enriched in lung SQCCs relative to adenocarcinomas, with four markers having prognostic significance in SQCCs. We also identified 33 signaling receptors, including the MST1R/RON growth factor receptor, whose ligand MST1/MSP was mitogenic for basal cells. This work provides the largest description to date of

Mixed cation FA x PEA 1- x PbI 3 with enhanced phase and ambient stability toward high-performance perovskite solar cells

DOE PAGES

Li, Nan; Zhu, Zonglong; Chueh, Chu -Chen; ...

2016-09-26

In this study, different from the commonly explored strategy of incorporating a smaller cation, MA + and Cs + into FAPbI 3 lattice to improve efficiency and stability, it is revealed that the introduction of phenylethylammonium iodide (PEAI) into FAPbI 3 perovksite to form mixed cation FA xPEA 1–xPbI 3 can effectively enhance both phase and ambient stability of FAPbI 3 as well as the resulting performance of the derived devices. From our experimental and theoretical calculation results, it is proposed that the larger PEA cation is capable of assembling on both the lattice surface and grain boundaries to formmore » quais-3D perovskite structures. The surrounding of PEA + ions at the crystal grain boundaries not only can serve as molecular locks to tighten FAPbI 3 domains but also passivate the surface defects to improve both phase and moisture stablity. Consequently, a high-performance (PCE:17.7%) and ambient stable FAPbI 3 solar cell could be developed« less

Folic acid conjugated cross-linked acrylic polymer (FA-CLAP) hydrogel for site specific delivery of hydrophobic drugs to cancer cells.

PubMed

Pillai, Jisha Jayadevan; Thulasidasan, Arun Kumar Theralikattu; Anto, Ruby John; Chithralekha, Devika Nandan; Narayanan, Ashwanikumar; Kumar, Gopalakrishnapillai Sankaramangalam Vinod

2014-07-15

The hydrogel based system is found to be rarely reported for the delivery of hydrophobic drug due to the incompatibility of hydrophilicity of the polymer network and the hydrophobicity of drug. This problem can be solved by preparing semi-interpenetrating network of cross-linked polymer for tuning the hydrophilicity so as to entrap the hydrophobic drugs. The current study is to develop a folic acid conjugated cross-linked pH sensitive, biocompatible polymeric hydrogel to achieve a site specific drug delivery. For that, we have synthesized a folic acid conjugated PEG cross-linked acrylic polymer (FA-CLAP) hydrogel and investigated its loading and release of curcumin. The formed polymer hydrogel was then conjugated with folic acid for the site specific delivery of curcumin to cancer cells and then further characterized and conducted the cell uptake and cytotoxicity studies on human cervical cancer cell lines (HeLa). In this study, we synthesized folic acid conjugated cross-linked acrylic hydrogel for the delivery of hydrophobic drugs to the cancer site. Poly (ethyleneglycol) (PEG) diacrylate cross-linked acrylic polymer (PAA) was prepared via inverse emulsion polymerization technique and later conjugated it with folic acid (FA-CLAP). Hydrophobic drug curcumin is entrapped into it and investigated the entrapment efficiency. Characterization of synthesized hydogel was done by using Fourier Transform-Infrared spectroscopy (FT-IR), Transmission Electron Microscopy (TEM), Differential Scanning Calorimetry (DSC). Polymerization and folate conjugation was confirmed by FT-IR spectroscopy. The release kinetics of drug from the entrapped form was studied which showed initial burst release followed by sustained release due to swelling and increased cross-linking. In vitro cytotoxicity and cell uptake studies were conducted in human cervical cancer (HeLa) cell lines. Results showed that curcumin entrapped folate conjugated cross-linked acrylic polymer (FA-CLAP) hydrogel showed

CRISPR/Cas9-Mediated Correction of the FANCD1 Gene in Primary Patient Cells.

PubMed

Skvarova Kramarzova, Karolina; Osborn, Mark J; Webber, Beau R; DeFeo, Anthony P; McElroy, Amber N; Kim, Chong Jai; Tolar, Jakub

2017-06-14

Fanconi anemia (FA) is an inherited condition characterized by impaired DNA repair, physical anomalies, bone marrow failure, and increased incidence of malignancy. Gene editing holds great potential to precisely correct the underlying genetic cause such that gene expression remains under the endogenous control mechanisms. This has been accomplished to date only in transformed cells or their reprogrammed induced pluripotent stem cell counterparts; however, it has not yet been reported in primary patient cells. Here we show the ability to correct a mutation in Fanconi anemia D1 ( FANCD1 ) primary patient fibroblasts. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was employed to target and correct a FANCD1 gene deletion. Homologous recombination using an oligonucleotide donor was achieved and a pure population of modified cells was obtained by using inhibitors of poly adenosine diphosphate-ribose polymerase (poly ADP-ribose polymerase). FANCD1 function was restored and we did not observe any promiscuous cutting of the CRISPR/Cas9 at off target sites. This consideration is crucial in the context of the pre-malignant FA phenotype. Altogether we show the ability to correct a patient mutation in primary FANCD1 cells in a precise manner. These proof of principle studies support expanded application of gene editing for FA.

Gremlin 1 Identifies a Skeletal Stem Cell with Bone, Cartilage, and Reticular Stromal Potential

PubMed Central

Worthley, Daniel L.; Churchill, Michael; Compton, Jocelyn T.; Tailor, Yagnesh; Rao, Meenakshi; Si, Yiling; Levin, Daniel; Schwartz, Matthew G.; Uygur, Aysu; Hayakawa, Yoku; Gross, Stefanie; Renz, Bernhard W.; Setlik, Wanda; Martinez, Ashley N.; Chen, Xiaowei; Nizami, Saqib; Lee, Heon Goo; Kang, H. Paco; Caldwell, Jon-Michael; Asfaha, Samuel; Westphalen, C. Benedikt; Graham, Trevor; Jin, Guangchun; Nagar, Karan; Wang, Hongshan; Kheirbek, Mazen A.; Kolhe, Alka; Carpenter, Jared; Glaire, Mark; Nair, Abhinav; Renders, Simon; Manieri, Nicholas; Muthupalani, Sureshkumar; Fox, James G.; Reichert, Maximilian; Giraud, Andrew S.; Schwabe, Robert F.; Pradere, Jean-Phillipe; Walton, Katherine; Prakash, Ajay; Gumucio, Deborah; Rustgi, Anil K.; Stappenbeck, Thaddeus S.; Friedman, Richard A.; Gershon, Michael D.; Sims, Peter; Grikscheit, Tracy; Lee, Francis Y.; Karsenty, Gerard; Mukherjee, Siddhartha; Wang, Timothy C.

2014-01-01

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs). PMID:25594183

Heterozygote FANCD2 mutations associated with childhood T Cell ALL and testicular seminoma.

PubMed

Smetsers, Stephanie; Muter, Joanne; Bristow, Claire; Patel, Leena; Chandler, Kate; Bonney, Denise; Wynn, Robert F; Whetton, Anthony D; Will, Andrew M; Rockx, Davy; Joenje, Hans; Strathdee, Gordon; Shanks, Jonathan; Klopocki, Eva; Gille, Johan J P; Dorsman, Josephine; Meyer, Stefan

2012-12-01

Fanconi anaemia (FA) is an inherited disease with congenital and developmental abnormalities characterised by cellular cross linker hypersensitivity. FA is caused by mutations in any of so far 15 identified FANC genes, which encode proteins that interact in a common DNA damage response (DDR) pathway. Individuals with FA have a high risk of developing acute myeloid leukaemia (AML) and squamous cell carcinoma. An increased cancer risk has been firmly established for carriers of mutations in FANCD1/BRCA2, FANCJ/BRIP1, FANCN/PALB2, RAD51C/FANCO and link the FA pathway to inherited breast and ovarian cancer. We describe a pedigree with FANCD2 mutations c.458T > C (p.Leu153Ser) and c.2715 + 1G > A (p.Glu906LeufsX4) with mild phenotype FA in the index case, T cell ALL in the Leu153Ser heterozygous brother and testicular seminoma in the p.Glu906LeufsX4 heterozygous father. Both FANCD2 alleles were present in the T Cell ALL and the seminoma. This links specific FANCD2 mutations to T cell ALL and seminoma without evidence of allelic loss in the tumour tissue.

Ferulic Acid Regulates the Nrf2/Heme Oxygenase-1 System and Counteracts Trimethyltin-Induced Neuronal Damage in the Human Neuroblastoma Cell Line SH-SY5Y

PubMed Central

Catino, Stefania; Paciello, Fabiola; Miceli, Fiorella; Rolesi, Rolando; Troiani, Diana; Calabrese, Vittorio; Santangelo, Rosaria; Mancuso, Cesare

2016-01-01

Over the past years, several lines of evidence have pointed out the efficacy of ferulic acid (FA) in counteracting oxidative stress elicited by β-amyloid or free radical initiators, based on the ability of this natural antioxidant to up-regulate the heme oxygenase-1 (HO-1) and biliverdin reductase (BVR) system. However, scarce results can be found in literature regarding the cytoprotective effects of FA in case of damage caused by neurotoxicants. The aim of this work is to investigate the mechanisms through which FA exerts neuroprotection in SH-SY5Y neuroblastoma cells exposed to the neurotoxin trimethyltin (TMT). FA (1–10 μM for 6 h) dose-dependently increased both basal and TMT (10 μM for 24 h)-induced HO-1 expression in SH-SY5Y cells by fostering the nuclear translocation of the transcriptional activator Nrf2. In particular, the co-treatment of FA (10 μM) with TMT was also responsible for the nuclear translocation of HO-1 in an attempt to further increase cell stress response in SH-SY5Y cells. In addition to HO-1, FA (1–10 μM for 6 h) dose-dependently increased the basal expression of BVR. The antioxidant and neuroprotective features of FA, through the increase of HO activity, were supported by the evidence that FA inhibited TMT (10 μM)-induced lipid peroxidation (evaluated by detecting 4-hydroxy-nonenal) and DNA fragmentation in SH-SY5Y cells and that this antioxidant effect was reversed by the HO inhibitor Zinc-protoporphyrin-IX (5 μM). Among the by-products of the HO/BVR system, carbon monoxide (CORM-2, 50 nM) and bilirubin (BR, 50 nM) significantly inhibited TMT-induced superoxide anion formation in SH-SY5Y cells. All together, these results corroborate the neuroprotective effect of FA through the up-regulation of the HO-1/BVR system, via carbon monoxide and BR formation, and provide the first evidence on the role of HO-1/Nrf2 axis in FA-related enhancement of cell stress response in human neurons. PMID:26779023

Mutational disruption of the ABCC2 gene in fall armyworm, Spodoptera frugiperda, confers resistance to the Cry1Fa and Cry1A.105 insecticidal proteins.

PubMed

Flagel, Lex; Lee, Young Wha; Wanjugi, Humphrey; Swarup, Shilpa; Brown, Alana; Wang, Jinling; Kraft, Edward; Greenplate, John; Simmons, Jeni; Adams, Nancy; Wang, Yanfei; Martinelli, Samuel; Haas, Jeffrey A; Gowda, Anilkumar; Head, Graham

2018-05-08

The use of Bt proteins in crops has revolutionized insect pest management by offering effective season-long control. However, field-evolved resistance to Bt proteins threatens their utility and durability. A recent example is field-evolved resistance to Cry1Fa and Cry1A.105 in fall armyworm (Spodoptera frugiperda). This resistance has been detected in Puerto Rico, mainland USA, and Brazil. A S. frugiperda population with suspected resistance to Cry1Fa was sampled from a maize field in Puerto Rico and used to develop a resistant lab colony. The colony demonstrated resistance to Cry1Fa and partial cross-resistance to Cry1A.105 in diet bioassays. Using genetic crosses and proteomics, we show that this resistance is due to loss-of-function mutations in the ABCC2 gene. We characterize two novel mutant alleles from Puerto Rico. We also find that these alleles are absent in a broad screen of partially resistant Brazilian populations. These findings confirm that ABCC2 is a receptor for Cry1Fa and Cry1A.105 in S. frugiperda, and lay the groundwork for genetically enabled resistance management in this species, with the caution that there may be several distinct ABCC2 resistances alleles in nature.

ABCB1 identifies a subpopulation of uveal melanoma cells with high metastatic propensity

PubMed Central

Landreville, Solange; Agapova, Olga A.; Kneass, Zachary T.; Salesse, Christian; Harbour, J. William

2011-01-01

SUMMARY Metastasis of tumor cells to distant organs is the leading cause of death in melanoma. Yet, the mechanisms of metastasis remain poorly understood. One key question is whether all cells in a primary tumor are equally likely to metastasize or whether subpopulations of cells preferentially give rise to metastases. Here, we identified a subpopulation of uveal melanoma cells expressing the multidrug resistance transporter ABCB1 that are highly metastatic compared to ABCB1− bulk tumor cells. ABCB1+ cells also exhibited enhanced clonogenicity, anchorage independent growth, tumorigenicity and mitochondrial activity compared to ABCB1− cells. A375 cutaneous melanoma cells contained a similar subpopulation of highly metastatic ABCB1+ cells. These findings suggest that some uveal melanoma cells have greater potential for metastasis than others, and that a better understanding of such cells may be necessary for more successful therapies for metastatic melanoma. PMID:21575142

Folic acid induces cell type-specific changes in the transcriptome of breast cancer cell lines: a proof-of-concept study.

PubMed

Price, R Jordan; Lillycrop, Karen A; Burdge, Graham C

2016-01-01

The effect of folic acid (FA) on breast cancer (BC) risk is uncertain. We hypothesised that this uncertainty may be due, in part, to differential effects of FA between BC cells with different phenotypes. To test this we investigated the effect of treatment with FA concentrations within the range of unmetabolised FA reported in humans on the expression of the transcriptome of non-transformed (MCF10A) and cancerous (MCF7 and Hs578T) BC cells. The total number of transcripts altered was: MCF10A, seventy-five (seventy up-regulated); MCF7, twenty-four (fourteen up-regulated); and Hs578T, 328 (156 up-regulated). Only the cancer-associated gene TAGLN was altered by FA in all three cell lines. In MCF10A and Hs578T cells, FA treatment decreased pathways associated with apoptosis, cell death and senescence, but increased those associated with cell proliferation. The folate transporters SLC19A1, SLC46A1 and FOLR1 were differentially expressed between cell lines tested. However, the level of expression was not altered by FA treatment. These findings suggest that physiological concentrations of FA can induce cell type-specific changes in gene regulation in a manner that is consistent with proliferative phenotype. This has implications for understanding the role of FA in BC risk. In addition, these findings support the suggestion that differences in gene expression induced by FA may involve differential activities of folate transporters. Together these findings indicate the need for further studies of the effect of FA on BC.

Far-red light photoacclimation (FaRLiP) in Synechococcus sp. PCC 7335. II.Characterization of phycobiliproteins produced during acclimation to far-red light.

PubMed

Ho, Ming-Yang; Gan, Fei; Shen, Gaozhong; Bryant, Donald A

2017-02-01

Phycobilisomes (PBS) are antenna complexes that harvest light for photosystem (PS) I and PS II in cyanobacteria and some algae. A process known as far-red light photoacclimation (FaRLiP) occurs when some cyanobacteria are grown in far-red light (FRL). They synthesize chlorophylls d and f and remodel PS I, PS II, and PBS using subunits paralogous to those produced in white light. The FaRLiP strain, Leptolyngbya sp. JSC-1, replaces hemidiscoidal PBS with pentacylindrical cores, which are produced when cells are grown in red or white light, with PBS with bicylindrical cores when cells are grown in FRL. This study shows that the PBS of another FaRLiP strain, Synechococcus sp. PCC 7335, are not remodeled in cells grown in FRL. Instead, cells grown in FRL produce bicylindrical cores that uniquely contain the paralogous allophycocyanin subunits encoded in the FaRLiP cluster, and these bicylindrical cores coexist with red-light-type PBS with tricylindrical cores. The bicylindrical cores have absorption maxima at 650 and 711 nm and a low-temperature fluorescence emission maximum at 730 nm. They contain ApcE2:ApcF:ApcD3:ApcD2:ApcD5:ApcB2 in the approximate ratio 2:2:4:6:12:22, and a structural model is proposed. Time course experiments showed that bicylindrical cores were detectable about 48 h after cells were transferred from RL to FRL and that synthesis of red-light-type PBS continued throughout a 21-day growth period. When considered in comparison with results for other FaRLiP cyanobacteria, the results here show that acclimation responses to FRL can differ considerably among FaRLiP cyanobacteria.

Chitosan-coated doxorubicin nano-particles drug delivery system inhibits cell growth of liver cancer via p53/PRC1 pathway.

PubMed

Ye, Bai-Liang; Zheng, Ru; Ruan, Xiao-Jiao; Zheng, Zhi-Hai; Cai, Hua-Jie

2018-01-01

Nano-particles have been widely used in target-specific drug delivery system and showed advantages in cancers treatment. This study aims to evaluate the effect of chitosan coated doxorubicin nano-particles drug delivery system in liver cancer. The chitosan nano-particles were prepared by using the ionic gelation method. The characterizations of the nano-particles were determined by transmission electron microscopy. The cytotoxicity was detected by MTT assay, and the endocytosis, cell apoptosis and cell cycle were examined by flow cytometry. The protein level was analyzed with western blot. The dual luciferase reporter assay was performed to assess the interaction between p53 and the promoter of PRC1, and chromatin immune-precipitation was used to verify the binding between them. The FA-CS-DOX nano-particles were irregular and spherical particles around 30-40 nm, with uniform size and no adhesion. No significant difference was noted in doxorubicin release rate between CS-DOX and FA-CS-DOX. FA-CS-DOX nano-particles showed stronger cytotoxicity than CS-DOX. FA-CS-DOX nano-particles promoted the apoptosis and arrested cell cycle at G2/M phase, and they up-regulated p53. FA-CS-DOX nano-particles inhibited cell survival through p53/PRC1 pathway. Chitosan-coated doxorubicin nano-particles drug delivery system inhibits cell growth of liver cancer by promoting apoptosis and arresting cell cycle at G2/M phase through p53/PRC1 pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

EspO1-2 Regulates EspM2-Mediated RhoA Activity to Stabilize Formation of Focal Adhesions in Enterohemorrhagic Escherichia coli-Infected Host Cells

PubMed Central

Iyoda, Sunao; Izumiya, Hidemasa; Watanabe, Haruo; Ohnishi, Makoto; Terajima, Jun

2013-01-01

Enterohemorrhagic Escherichia coli (EHEC) Sakai strain encodes two homologous type III effectors, EspO1-1 and EspO1-2. These EspO1s have amino acid sequence homology with Shigella OspE, which targets integrin-linked kinase to stabilize formation of focal adhesions (FAs). Like OspE, EspO1-1 was localized to FAs in EHEC-infected cells, but EspO1-2 was localized in the cytoplasm. An EHEC ΔespO1-1ΔespO1-2 double mutant induced cell rounding and FA loss in most of infected cells, but neither the ΔespO1-1 nor ΔespO1-2 single mutant did. These results suggested that EspO1-2 functioned in the cytoplasm by a different mechanism from EspO1-1 and OspE. Since several type III effectors modulate Rho GTPase, which contributes to FA formation, we investigated whether EspO1-2 modulates the function of these type III effectors. We identified a direct interaction between EspO1-2 and EspM2, which acts as a RhoA guanine nucleotide exchange factor. Upon ectopic co-expression, EspO1-2 co-localized with EspM2 in the cytoplasm and suppressed EspM2-mediated stress fiber formation. Consistent with these findings, an ΔespO1-1ΔespO1-2ΔespM2 triple mutant did not induce cell rounding in epithelial cells. These results indicated that EspO1-2 interacted with EspM2 to regulate EspM2-mediated RhoA activity and stabilize FA formation during EHEC infection. PMID:23409096

Opuntia ficus indica (nopal) attenuates hepatic steatosis and oxidative stress in obese Zucker (fa/fa) rats.

PubMed

Morán-Ramos, Sofía; Avila-Nava, Azalia; Tovar, Armando R; Pedraza-Chaverri, José; López-Romero, Patricia; Torres, Nimbe

2012-11-01

Nonalcoholic fatty liver disease (NAFLD) is associated with multiple factors such as obesity, insulin resistance, and oxidative stress. Nopal, a cactus plant widely consumed in the Mexican diet, is considered a functional food because of its antioxidant activity and ability to improve biomarkers of metabolic syndrome. The aim of this study was to assess the effect of nopal consumption on the development of hepatic steatosis and hepatic oxidative stress and on the regulation of genes involved in hepatic lipid metabolism. Obese Zucker (fa/fa) rats were fed a control diet or a diet containing 4% nopal for 7 wk. Rats fed the nopal-containing diet had ∼50% lower hepatic TG than the control group as well as a reduction in hepatomegaly and biomarkers of hepatocyte injury such as alanine and aspartate aminotransferases. Attenuation of hepatic steatosis by nopal consumption was accompanied by a higher serum concentration of adiponectin and a greater abundance of mRNA for genes involved in lipid oxidation and lipid export and production of carnitine palmitoyltransferase-1 and microsomal TG transfer proteins in liver. Hepatic reactive oxygen species and lipid peroxidation biomarkers were significantly lower in rats fed nopal compared with the control rats. Furthermore, rats fed the nopal diet had a lower postprandial serum insulin concentration and a greater liver phosphorylated protein kinase B (pAKT):AKT ratio in the postprandial state. This study suggests that nopal consumption attenuates hepatic steatosis by increasing fatty acid oxidation and VLDL synthesis, decreasing oxidative stress, and improving liver insulin signaling in obese Zucker (fa/fa) rats.

Effects of antiglucocorticoid RU 486 on development of obesity in obese fa/fa Zucker rats.

PubMed

Langley, S C; York, D A

1990-09-01

The effects of RU 486 (mitepristone), an antagonist of type II glucocorticoid receptors (GR), on the development of obesity in young 5-wk-old obese fa/fa rats has been investigated. After 15 days of treatment, body composition of obese RU 486-treated rats was similar to that of lean-vehicle rats. Analysis of body composition changes showed that RU 486 effectively reversed the obesity. It stopped fat deposition in obese rats but increased protein deposition to the level of lean-vehicle rats. RU 486 prevented the development of hyperphagia and reduced gross energetic efficiency in the obese rats but had little effect on lean rats. Brown adipose tissue mitochondrial GDP binding was increased in obese rats but was reduced in lean rats by RU 486 treatment. RU 486 also reduced the elevated activity of hippocampal glycerophosphate dehydrogenase, a glucocorticoid-responsive enzyme, of obese rats to the level of lean rats. The evidence suggests that abnormal activity of glucocorticoid GR receptors or abnormal cellular responsiveness to corticosterone receptor complexes may be important in the development of obesity in the fa/fa rat.

The dam replacing gene product enhances Neisseria gonorrhoeae FA1090 viability and biofilm formation

PubMed Central

Kwiatek, Agnieszka; Bacal, Pawel; Wasiluk, Adrian; Trybunko, Anastasiya; Adamczyk-Poplawska, Monika

2014-01-01

Many Neisseriaceae do not exhibit Dam methyltransferase activity and, instead of the dam gene, possess drg (dam replacing gene) inserted in the leuS/dam locus. The drg locus in Neisseria gonorrhoeae FA1090 has a lower GC-pairs content (40.5%) compared to the whole genome of N. gonorrhoeae FA1090 (52%). The gonococcal drg gene encodes a DNA endonuclease Drg, with GmeATC specificity. Disruption of drg or insertion of the dam gene in gonococcal genome changes the level of expression of genes as shown by transcriptome analysis. For the drg-deficient N. gonorrhoeae mutant, a total of 195 (8.94% of the total gene pool) genes exhibited an altered expression compared to the wt strain by at least 1.5 fold. In dam-expressing N. gonorrhoeae mutant, the expression of 240 genes (11% of total genes) was deregulated. Most of these deregulated genes were involved in translation, DNA repair, membrane biogenesis and energy production as shown by cluster of orthologous group analysis. In vivo, the inactivation of drg gene causes the decrease of the number of live neisserial cells and long lag phase of growth. The insertion of dam gene instead of drg locus restores cell viability. We have also shown that presence of the drg gene product is important for N. gonorrhoeae FA1090 in adhesion, including human epithelial cells, and biofilm formation. Biofilm produced by drg-deficient strain is formed by more dispersed cells, compared to this one formed by parental strain as shown by scanning electron and confocal microscopy. Also adherence assays show a significantly smaller biomass of formed biofilm (OD570 = 0.242 ± 0.038) for drg-deficient strain, compared to wild-type strain (OD570 = 0.378 ± 0.057). Dam-expressing gonococcal cells produce slightly weaker biofilm with cells embedded in an extracellular matrix. This strain has also a five times reduced ability for adhesion to human epithelial cells. In this context, the presence of Drg is more advantageous for N. gonorrhoeae biology than

JPRS Report Science & Technology Japan Future Prospects of FA-From FA to IMS

DTIC Science & Technology

1989-12-04

of FA Investment (Large Companies) a) m d) m 5000fI~ljl|S 1 m± ¥-1$ 500~1000t| 300~500(f 1000~3000ft i) £ÜÜ 0i!§£3^ SWOT # H^fc3S j) k) 1...is a method conceived by Toyota Motors, now widely understood throughout the world. Such information as the production volume, time, method and...T. Suzuki Toyota Motor Corp. Y. Tatsue AIST, Mechanical Engineering Laboratory K. Togino Komatsu Ltd. H. Torii Nihon Keizai Shimbun Editorial

Supplemental fructose attenuates postprandial glycemia in Zucker fatty fa/fa rats.

PubMed

Wolf, Bryan W; Humphrey, Phillip M; Hadley, Craig W; Maharry, Kati S; Garleb, Keith A; Firkins, Jeffrey L

2002-06-01

Experiments were conducted to evaluate the effects of supplemental fructose on postprandial glycemia. After overnight food deprivation, Zucker fatty fa/fa rats were given a meal glucose tolerance test. Plasma glucose response was determined for 180 min postprandially. At a dose of 0.16 g/kg body, fructose reduced (P < 0.05) the incremental area under the curve (AUC) by 34% when supplemented to a glucose challenge and by 32% when supplemented to a maltodextrin (a rapidly digested starch) challenge. Similarly, sucrose reduced (P = 0.0575) the incremental AUC for plasma glucose when rats were challenged with maltodextrin. Second-meal glycemic response was not affected by fructose supplementation to the first meal, and fructose supplementation to the second meal reduced (P < 0.05) postprandial glycemia when fructose had been supplemented to the first meal. In a dose-response study (0.1, 0.2, and 0.5 g/kg body), supplemental fructose reduced (P < 0.01) the peak rise in plasma glucose (linear and quadratic effects). In the final experiment, a low dose of fructose (0.075 g/kg body) reduced (P < 0.05) the incremental AUC by 18%. These data support the hypothesis that small amounts of oral fructose or sucrose may be useful in lowering the postprandial blood glucose response.

Tumor cell migration screen identifies SRPK1 as breast cancer metastasis determinant

PubMed Central

van Roosmalen, Wies; Le Dévédec, Sylvia E.; Golani, Ofra; Smid, Marcel; Pulyakhina, Irina; Timmermans, Annemieke M.; Look, Maxime P.; Zi, Di; Pont, Chantal; de Graauw, Marjo; Naffar-Abu-Amara, Suha; Kirsanova, Catherine; Rustici, Gabriella; Hoen, Peter A.C. ‘t; Martens, John W.M.; Foekens, John A.; Geiger, Benjamin; van de Water, Bob

2015-01-01

Tumor cell migration is a key process for cancer cell dissemination and metastasis that is controlled by signal-mediated cytoskeletal and cell matrix adhesion remodeling. Using a phagokinetic track assay with migratory H1299 cells, we performed an siRNA screen of almost 1,500 genes encoding kinases/phosphatases and adhesome- and migration-related proteins to identify genes that affect tumor cell migration speed and persistence. Thirty candidate genes that altered cell migration were validated in live tumor cell migration assays. Eight were associated with metastasis-free survival in breast cancer patients, with integrin β3–binding protein (ITGB3BP), MAP3K8, NIMA-related kinase (NEK2), and SHC-transforming protein 1 (SHC1) being the most predictive. Examination of genes that modulate migration indicated that SRPK1, encoding the splicing factor kinase SRSF protein kinase 1, is relevant to breast cancer outcomes, as it was highly expressed in basal breast cancer. Furthermore, high SRPK1 expression correlated with poor breast cancer disease outcome and preferential metastasis to the lungs and brain. In 2 independent murine models of breast tumor metastasis, stable shRNA-based SRPK1 knockdown suppressed metastasis to distant organs, including lung, liver, and spleen, and inhibited focal adhesion reorganization. Our study provides comprehensive information on the molecular determinants of tumor cell migration and suggests that SRPK1 has potential as a drug target for limiting breast cancer metastasis. PMID:25774502

Tumor cell migration screen identifies SRPK1 as breast cancer metastasis determinant.

PubMed

van Roosmalen, Wies; Le Dévédec, Sylvia E; Golani, Ofra; Smid, Marcel; Pulyakhina, Irina; Timmermans, Annemieke M; Look, Maxime P; Zi, Di; Pont, Chantal; de Graauw, Marjo; Naffar-Abu-Amara, Suha; Kirsanova, Catherine; Rustici, Gabriella; Hoen, Peter A C 't; Martens, John W M; Foekens, John A; Geiger, Benjamin; van de Water, Bob

2015-04-01

Tumor cell migration is a key process for cancer cell dissemination and metastasis that is controlled by signal-mediated cytoskeletal and cell matrix adhesion remodeling. Using a phagokinetic track assay with migratory H1299 cells, we performed an siRNA screen of almost 1,500 genes encoding kinases/phosphatases and adhesome- and migration-related proteins to identify genes that affect tumor cell migration speed and persistence. Thirty candidate genes that altered cell migration were validated in live tumor cell migration assays. Eight were associated with metastasis-free survival in breast cancer patients, with integrin β3-binding protein (ITGB3BP), MAP3K8, NIMA-related kinase (NEK2), and SHC-transforming protein 1 (SHC1) being the most predictive. Examination of genes that modulate migration indicated that SRPK1, encoding the splicing factor kinase SRSF protein kinase 1, is relevant to breast cancer outcomes, as it was highly expressed in basal breast cancer. Furthermore, high SRPK1 expression correlated with poor breast cancer disease outcome and preferential metastasis to the lungs and brain. In 2 independent murine models of breast tumor metastasis, stable shRNA-based SRPK1 knockdown suppressed metastasis to distant organs, including lung, liver, and spleen, and inhibited focal adhesion reorganization. Our study provides comprehensive information on the molecular determinants of tumor cell migration and suggests that SRPK1 has potential as a drug target for limiting breast cancer metastasis.

A genome-wide association study of red-blood cell fatty acids and ratios incorporating dietary covariates: Framingham Heart Study Offspring Cohort.

PubMed

Kalsbeek, Anya; Veenstra, Jenna; Westra, Jason; Disselkoen, Craig; Koch, Kristin; McKenzie, Katelyn A; O'Bott, Jacob; Vander Woude, Jason; Fischer, Karen; Shearer, Greg C; Harris, William S; Tintle, Nathan L

2018-01-01

Recent analyses have suggested a strong heritable component to circulating fatty acid (FA) levels; however, only a limited number of genes have been identified which associate with FA levels. In order to expand upon a previous genome wide association study done on participants in the Framingham Heart Study Offspring Cohort and FA levels, we used data from 2,400 of these individuals for whom red blood cell FA profiles, dietary information and genotypes are available, and then conducted a genome-wide evaluation of potential genetic variants associated with 22 FAs and 15 FA ratios, after adjusting for relevant dietary covariates. Our analysis found nine previously identified loci associated with FA levels (FADS, ELOVL2, PCOLCE2, LPCAT3, AGPAT4, NTAN1/PDXDC1, PKD2L1, HBS1L/MYB and RAB3GAP1/MCM6), while identifying four novel loci. The latter include an association between variants in CALN1 (Chromosome 7) and eicosapentaenoic acid (EPA), DHRS4L2 (Chromosome 14) and a FA ratio measuring delta-9-desaturase activity, as well as two loci associated with less well understood proteins. Thus, the inclusion of dietary covariates had a modest impact, helping to uncover four additional loci. While genome-wide association studies continue to uncover additional genes associated with circulating FA levels, much of the heritable risk is yet to be explained, suggesting the potential role of rare genetic variation, epistasis and gene-environment interactions on FA levels as well. Further studies are needed to continue to understand the complex genetic picture of FA metabolism and synthesis.

Friedreich's Ataxia (FA)

MedlinePlus

... success- ful people with FA — business leaders, outstanding students, engineers, active teens and bright kids, parents, even ... tory experiments have shown that it’s possible to design short fragments of DNA that prevent abnormal folding ...

The immune receptor Trem1 cooperates with diminished DNA damage response to induce preleukemic stem cell expansion.

PubMed

Du, W; Amarachintha, S; Wilson, A; Pang, Q

2017-02-01

Fanconi anemia (FA) is an inherited bone marrow failure syndrome with extremely high risk of leukemic transformation. Here we investigate the relationship between DNA damage response (DDR) and leukemogenesis using the Fanca knockout mouse model. We found that chronic exposure of the Fanca -/- hematopoietic stem cells to DNA crosslinking agent mitomycin C in vivo leads to diminished DDR, and the emergence/expansion of pre-leukemia stem cells (pre-LSCs). Surprisingly, although genetic correction of Fanca deficiency in the pre-LSCs restores DDR and reduces genomic instability, but fails to prevent pre-LSC expansion or delay leukemia development in irradiated recipients. Furthermore, we identified transcription program underlying dysregulated DDR and cell migration, myeloid proliferation, and immune response in the Fanca -/- pre-LSCs. Forced expression of the downregulated DNA repair genes, Rad51c or Trp53i13, in the Fanca -/- pre-LSCs partially rescues DDR but has no effect on leukemia, whereas shRNA knockdown of the upregulated immune receptor genes Trem1 or Pilrb improves leukemia-related survival, but not DDR or genomic instability. Furthermore, Trem1 cooperates with diminished DDR in vivo to promote Fanca -/- pre-LSC expansion and leukemia development. Our study implicates diminishing DDR as a root cause of FA leukemogenesis, which subsequently collaborates with other signaling pathways for leukemogenic transformation.

Formal language of Lanna Shop House’s Façade in Lampang Old city, Thailand

NASA Astrophysics Data System (ADS)

Phetsuriya, Natthakit

2017-10-01

This article aims to presents ‘the formal architectural language of Lanna Designs” that is a linguistic paradigm for decrypt the linguistic system which is hidden in the Lanna façade style. Lanna Designs present an identity of vital ordered and crucial articulated formal language which inherently set of mathematical rules for the arrangement of ornaments. The scope of this article is attempted to the morphology of façades of the ten shop houses which located in Lampang Old city and have familiar proportion and style. In this article, the sampling of façade buildings required proportion as three-stall and two-story with familiar style. The morphology is described based on terms of a symbolic encoding system that is represented as graphically building grammar. The system helps to emphasize commonalities in façade languages and propose a prototype of identified Lanna façade design. This methodology might be the option for decrypt or study in every facades style.

Cosmologically allowed regions for the axion decay constant Fa

NASA Astrophysics Data System (ADS)

Kawasaki, Masahiro; Sonomoto, Eisuke; Yanagida, Tsutomu T.

2018-07-01

If the Peccei-Quinn symmetry is already broken during inflation, the decay constant Fa of the axion can be in a wide region from 1011GeV to 1018GeV for the axion being the dominant dark matter. In this case, however, the axion causes the serious cosmological problem, isocurvature perturbation problem, which severely constrains the Hubble parameter during inflation. The constraint is relaxed when Peccei-Quinn scalar field takes a large value ∼Mp (Planck scale) during inflation. In this letter, we point out that the allowed region of the decay constant Fa is reduced to a rather narrow region for a given tensor-to-scalar ratio r when Peccei-Quinn scalar field takes ∼Mp during inflation. For example, if the ratio r is determined as r ≳10-3 in future measurements, we can predict Fa ≃ (0.1- 1.4) ×1012GeV for domain wall number NDW = 6.

Stem cell gene therapy for fanconi anemia: report from the 1st international Fanconi anemia gene therapy working group meeting.

PubMed

Tolar, Jakub; Adair, Jennifer E; Antoniou, Michael; Bartholomae, Cynthia C; Becker, Pamela S; Blazar, Bruce R; Bueren, Juan; Carroll, Thomas; Cavazzana-Calvo, Marina; Clapp, D Wade; Dalgleish, Robert; Galy, Anne; Gaspar, H Bobby; Hanenberg, Helmut; Von Kalle, Christof; Kiem, Hans-Peter; Lindeman, Dirk; Naldini, Luigi; Navarro, Susana; Renella, Raffaele; Rio, Paula; Sevilla, Julián; Schmidt, Manfred; Verhoeyen, Els; Wagner, John E; Williams, David A; Thrasher, Adrian J

2011-07-01

Survival rates after allogeneic hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) have increased dramatically since 2000. However, the use of autologous stem cell gene therapy, whereby the patient's own blood stem cells are modified to express the wild-type gene product, could potentially avoid the early and late complications of allogeneic HCT. Over the last decades, gene therapy has experienced a high degree of optimism interrupted by periods of diminished expectation. Optimism stems from recent examples of successful gene correction in several congenital immunodeficiencies, whereas diminished expectations come from the realization that gene therapy will not be free of side effects. The goal of the 1st International Fanconi Anemia Gene Therapy Working Group Meeting was to determine the optimal strategy for moving stem cell gene therapy into clinical trials for individuals with FA. To this end, key investigators examined vector design, transduction method, criteria for large-scale clinical-grade vector manufacture, hematopoietic cell preparation, and eligibility criteria for FA patients most likely to benefit. The report summarizes the roadmap for the development of gene therapy for FA.

Stem Cell Gene Therapy for Fanconi Anemia: Report from the 1st International Fanconi Anemia Gene Therapy Working Group Meeting

PubMed Central

Tolar, Jakub; Adair, Jennifer E; Antoniou, Michael; Bartholomae, Cynthia C; Becker, Pamela S; Blazar, Bruce R; Bueren, Juan; Carroll, Thomas; Cavazzana-Calvo, Marina; Clapp, D Wade; Dalgleish, Robert; Galy, Anne; Gaspar, H Bobby; Hanenberg, Helmut; Von Kalle, Christof; Kiem, Hans-Peter; Lindeman, Dirk; Naldini, Luigi; Navarro, Susana; Renella, Raffaele; Rio, Paula; Sevilla, Julián; Schmidt, Manfred; Verhoeyen, Els; Wagner, John E; Williams, David A; Thrasher, Adrian J

2011-01-01

Survival rates after allogeneic hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) have increased dramatically since 2000. However, the use of autologous stem cell gene therapy, whereby the patient's own blood stem cells are modified to express the wild-type gene product, could potentially avoid the early and late complications of allogeneic HCT. Over the last decades, gene therapy has experienced a high degree of optimism interrupted by periods of diminished expectation. Optimism stems from recent examples of successful gene correction in several congenital immunodeficiencies, whereas diminished expectations come from the realization that gene therapy will not be free of side effects. The goal of the 1st International Fanconi Anemia Gene Therapy Working Group Meeting was to determine the optimal strategy for moving stem cell gene therapy into clinical trials for individuals with FA. To this end, key investigators examined vector design, transduction method, criteria for large-scale clinical-grade vector manufacture, hematopoietic cell preparation, and eligibility criteria for FA patients most likely to benefit. The report summarizes the roadmap for the development of gene therapy for FA. PMID:21540837

Effect of combination treatment of S–amlodipine with peroxisome proliferator-activated receptor agonists on metabolic and cardiovascular parameters in Zucker fa/fa rats

PubMed Central

2014-01-01

Background Type 2 diabetes is a complex metabolic disorder characterized by hyperglycemia, impaired glucose tolerance and insulin resistance associated with dyslipidemia and hypertension. The available drugs are not sufficiently efficacious in reducing cardiovascular risk and restoring normal glucose metabolism associated with type 2 diabetes as a mono- or a combination therapy. The present study examined the combined effects of an antihypertensive (S-Amlodipine) and an insulin-sensitizing agent, peroxisome proliferator-activated receptor (PPAR) agonists (Pioglitazone and Ragaglitazar), on cardiovascular risk factors in aged diabetic and insulin-resistant Zucker fa/fa rats. Methods Following combination treatment for 14 days, blood pressure (BP), serum glucose, total cholesterol and triglycerides were measured. Aortic ring study was conducted to determine the effect of combination treatments on phenylephrine-induced vasoconstriction and acetylcholine (Ach)-induced vasorelaxation. Results In combination, S-Amlodipine and Pioglitazone significantly reduced blood glucose (115.1 ± 6.6 vs. 81.7 ± 4.2), BP (184.4 ± 5.0 vs. 155.1 ± 5.0), serum triglycerides (362.5 ± 47.5 vs. 211.1 ± 23.7) and glucose intolerance when compared with vehicle treated Zucker fa/fa rats. Similar results were observed with the combination of S-Amlodipine and Ragaglitazar (Triglycerides, 362.5 ± 47.5 vs. 252.34 ± 27.86; BP, 184.4 ± 5.0 vs. 159.0 ± 8.0) except for serum glucose. ACh-induced vasorelaxation in aortic rings was also superior with both of the combinations compared to individual treatment. Furthermore, there was less body weight gain and food intake with S-Amlodipine and Pioglitazone combination in Zucker fa/fa rats. S-Amlodipine itself caused significant reduction in glucose (115.1 ± 6.6 vs. 89.7 ± 2.7) and BP (184.4 ± 5.0 vs. 156.1 ± 4.0) with improvement in insulin sensitivity observed through oral glucose

This NASA Dryden F/A-18 is participating in the Automated Aerial Refueling (AAR) project. F/A-18 (No

NASA Technical Reports Server (NTRS)

2002-01-01

A NASA Dryden F/A-18 is participating in the Automated Aerial Refueling (AAR) project. F/A-18 (No. 847) is acting as an in-flight refueling tanker in the study to develop analytical models for an automated aerial refueling system for unmanned vehicles. A 300-gallon aerodynamic pod containing air-refueling equipment is seen beneath the fuselage. The hose and refueling basket are extended during an assessment of their dynamics on the F/A-18A.

Reduced Hepatic Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 Level in Obesity.

PubMed

Heinrich, Garrett; Muturi, Harrison T; Rezaei, Khadijeh; Al-Share, Qusai Y; DeAngelis, Anthony M; Bowman, Thomas A; Ghadieh, Hilda E; Ghanem, Simona S; Zhang, Deqiang; Garofalo, Robert S; Yin, Lei; Najjar, Sonia M

2017-01-01

Impairment of insulin clearance is being increasingly recognized as a critical step in the development of insulin resistance and metabolic disease. The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes insulin clearance. Null deletion or liver-specific inactivation of Ceacam1 in mice causes a defect in insulin clearance, insulin resistance, steatohepatitis, and visceral obesity. Immunohistological analysis revealed reduction of hepatic CEACAM1 in obese subjects with fatty liver disease. Thus, we aimed to determine whether this occurs at the hepatocyte level in response to systemic extrahepatic factors and whether this holds across species. Northern and Western blot analyses demonstrate that CEACAM1 mRNA and protein levels are reduced in liver tissues of obese individuals compared to their lean age-matched counterparts. Furthermore, Western analysis reveals a comparable reduction of CEACAM1 protein in primary hepatocytes derived from the same obese subjects. Similar to humans, Ceacam1 mRNA level, assessed by quantitative RT-PCR analysis, is significantly reduced in the livers of obese Zucker ( fa/fa , ZDF) and Koletsky ( f/f ) rats relative to their age-matched lean counterparts. These studies demonstrate that the reduction of hepatic CEACAM1 in obesity occurs at the level of hepatocytes and identify the reduction of hepatic CEACAM1 as a common denominator of obesity across multiple species.

Diets containing salmon fillet delay development of high blood pressure and hyperfusion damage in kidneys in obese Zucker fa/fa rats.

PubMed

Vikøren, Linn A; Drotningsvik, Aslaug; Mwakimonga, Angela; Leh, Sabine; Mellgren, Gunnar; Gudbrandsen, Oddrun A

2018-04-01

Hypertension is the leading risk factor for cardiovascular and chronic renal diseases, affecting more than 1 billion people. Fish intake is inversely correlated with the prevalence of hypertension in several, but not all, studies, and intake of fish oil and fish proteins has shown promising potential to delay development of high blood pressure in rats. The effects of baked and raw salmon fillet intake on blood pressure and renal function were investigated in obese Zucker fa/fa rats, which spontaneously develop hypertension with proteinuria and renal failure. Rats were fed diets containing baked or raw salmon fillet in an amount corresponding to 25% of total protein from salmon and 75% of protein from casein, or casein as the sole protein source (control group) for 4 weeks. Results show lower blood pressure and lower urine concentrations of albumin and cystatin C (relative to creatinine) in salmon diet groups when compared to control group. Morphological examinations revealed less prominent hyperfusion damage in podocytes from rats fed diets containing baked or raw salmon when compared to control rats. In conclusion, diets containing baked or raw salmon fillet delayed the development of hypertension and protected against podocyte damage in obese Zucker fa/fa rats. Copyright © 2018 American Heart Association. Published by Elsevier Inc. All rights reserved.

Gonadotropin-Releasing Hormone Regulates Expression of the DNA Damage Repair Gene, Fanconi anemia A, in Pituitary Gonadotroph Cells1

PubMed Central

Larder, Rachel; Chang, Lynda; Clinton, Michael; Brown, Pamela

2007-01-01

Gonadal function is critically dependant on regulated secretion of the gonadotropin hormones from anterior pituitary gonadotroph cells. Gonadotropin biosynthesis and release is triggered by the binding of hypothalamic GnRH to GnRH receptor expressed on the gonadotroph cell surface. The repertoire of regulatory molecules involved in this process are still being defined. We used the mouse LβT2 gonadotroph cell line, which expresses both gonadotropin hormones, as a model to investigate GnRH regulation of gene expression and differential display reverse transcription-polymerase chain reaction (RT-PCR) to identify and isolate hormonally induced changes. This approach identified Fanconi anemia a (Fanca), a gene implicated in DNA damage repair, as a differentially expressed transcript. Mutations in Fanca account for the majority of cases of Fanconi anemia (FA), a recessively inherited disease identified by congenital defects, bone marrow failure, infertility, and cancer susceptibility. We confirmed expression and hormonal regulation of Fanca mRNA by quantitative RT-PCR, which showed that GnRH induced a rapid, transient increase in Fanca mRNA. Fanca protein was also acutely upregulated after GnRH treatment of LβT2 cells. In addition, Fanca gene expression was confined to mature pituitary gonadotrophs and adult mouse pituitary and was not expressed in the immature αT3-1 gonadotroph cell line. Thus, this study extends the expression profile of Fanca into a highly specialized endocrine cell and demonstrates hormonal regulation of expression of the Fanca locus. We suggest that this regulatory mechanism may have a crucial role in the GnRH-response mechanism of mature gonadotrophs and perhaps the etiology of FA. PMID:15128600

A genome-wide association study of red-blood cell fatty acids and ratios incorporating dietary covariates: Framingham Heart Study Offspring Cohort

PubMed Central

Veenstra, Jenna; Westra, Jason; Disselkoen, Craig; Koch, Kristin; McKenzie, Katelyn A.; O’Bott, Jacob; Vander Woude, Jason; Fischer, Karen; Shearer, Greg C.; Harris, William S.; Tintle, Nathan L.

2018-01-01

Recent analyses have suggested a strong heritable component to circulating fatty acid (FA) levels; however, only a limited number of genes have been identified which associate with FA levels. In order to expand upon a previous genome wide association study done on participants in the Framingham Heart Study Offspring Cohort and FA levels, we used data from 2,400 of these individuals for whom red blood cell FA profiles, dietary information and genotypes are available, and then conducted a genome-wide evaluation of potential genetic variants associated with 22 FAs and 15 FA ratios, after adjusting for relevant dietary covariates. Our analysis found nine previously identified loci associated with FA levels (FADS, ELOVL2, PCOLCE2, LPCAT3, AGPAT4, NTAN1/PDXDC1, PKD2L1, HBS1L/MYB and RAB3GAP1/MCM6), while identifying four novel loci. The latter include an association between variants in CALN1 (Chromosome 7) and eicosapentaenoic acid (EPA), DHRS4L2 (Chromosome 14) and a FA ratio measuring delta-9-desaturase activity, as well as two loci associated with less well understood proteins. Thus, the inclusion of dietary covariates had a modest impact, helping to uncover four additional loci. While genome-wide association studies continue to uncover additional genes associated with circulating FA levels, much of the heritable risk is yet to be explained, suggesting the potential role of rare genetic variation, epistasis and gene-environment interactions on FA levels as well. Further studies are needed to continue to understand the complex genetic picture of FA metabolism and synthesis. PMID:29652918

FANCG promotes formation of a newly identified protein complex containing BRCA2, FANCD2 and XRCC3.

PubMed

Wilson, J B; Yamamoto, K; Marriott, A S; Hussain, S; Sung, P; Hoatlin, M E; Mathew, C G; Takata, M; Thompson, L H; Kupfer, G M; Jones, N J

2008-06-12

Fanconi anemia (FA) is a human disorder characterized by cancer susceptibility and cellular sensitivity to DNA crosslinks and other damages. Thirteen complementation groups and genes are identified, including BRCA2, which is defective in the FA-D1 group. Eight of the FA proteins, including FANCG, participate in a nuclear core complex that is required for the monoubiquitylation of FANCD2 and FANCI. FANCD2, like FANCD1/BRCA2, is not part of the core complex, and we previously showed direct BRCA2-FANCD2 interaction using yeast two-hybrid analysis. We now show in human and hamster cells that expression of FANCG protein, but not the other core complex proteins, is required for co-precipitation of BRCA2 and FANCD2. We also show that phosphorylation of FANCG serine 7 is required for its co-precipitation with BRCA2, XRCC3 and FANCD2, as well as the direct interaction of BRCA2-FANCD2. These results argue that FANCG has a role independent of the FA core complex, and we propose that phosphorylation of serine 7 is the signalling event required for forming a discrete complex comprising FANCD1/BRCA2-FANCD2-FANCG-XRCC3 (D1-D2-G-X3). Cells that fail to express either phospho-Ser7-FANCG, or full length BRCA2 protein, lack the interactions amongst the four component proteins. A role for D1-D2-G-X3 in homologous recombination repair (HRR) is supported by our finding that FANCG and the RAD51-paralog XRCC3 are epistatic for sensitivity to DNA crosslinking compounds in DT40 chicken cells. Our findings further define the intricate interface between FANC and HRR proteins in maintaining chromosome stability.

NCR1 Expression Identifies Canine Natural Killer Cell Subsets with Phenotypic Similarity to Human Natural Killer Cells

PubMed Central

Foltz, Jennifer A.; Somanchi, Srinivas S.; Yang, Yanwen; Aquino-Lopez, Arianexys; Bishop, Erin E.; Lee, Dean A.

2016-01-01

Canines spontaneously develop many cancers similar to humans – including osteosarcoma, leukemia, and lymphoma – offering the opportunity to study immune therapies in a genetically heterogeneous and immunocompetent environment. However, a lack of antibodies recognizing canine NK cell markers has resulted in suboptimal characterization and unknown purity of NK cell products, hindering the development of canine models of NK cell adoptive immunotherapy. To this end, we generated a novel antibody to canine NCR1 (NKp46), the putative species-wide marker of NK cells, enabling purification of NK cells for further characterization. We demonstrate that CD3−/NKp46+ cells in healthy and osteosarcoma-bearing canines have phenotypic similarity to human CD3−/NKp46+ NK cells, expressing mRNA for CD16 and the natural cytotoxicity receptors NKp30, NKp44, and NKp80. Functionally, we demonstrate with the calcein release assay that canine CD3−/NKp46+ cells kill canine tumor cell lines without prior sensitization and secrete IFN-γ, TNF-α, IL-8, IL-10, and granulocyte-macrophage colony-stimulating factor as measured by Luminex. Similar to human NK cells, CD3−/NKp46+ cells expand rapidly on feeder cells expressing 4-1BBL and membrane-bound IL-21 (median = 20,283-fold in 21 days). Furthermore, we identify a minor Null population (CD3−/CD21−/CD14−/NKp46−) with reduced cytotoxicity against osteosarcoma cells, but similar cytokine secretion as CD3−/NKp46+ cells. Null cells in canines and humans have reduced expression of NKG2D, NKp44, and CD16 compared to NKp46+ NK cells and can be induced to express NKp46 with further expansion on feeder cells. In conclusion, we have identified and characterized canine NK cells, including an NKp46− subset of canine and human NK cells, using a novel anti-canine NKp46 antibody, and report robust ex vivo expansion of canine NK cells sufficient for adoptive immunotherapy. PMID:27933061

Lipidomic Profiling Links the Fanconi Anemia Pathway to Glycosphingolipid Metabolism in Head and Neck Cancer Cells.

PubMed

Zhao, Xueheng; Brusadelli, Marion G; Sauter, Sharon; Butsch Kovacic, Melinda; Zhang, Wujuan; Romick-Rosendale, Lindsey E; Lambert, Paul F; Setchell, Kenneth D R; Wells, Susanne I

2018-06-01

Purpose: Mutations in Fanconi anemia (FA) genes are common in sporadic squamous cell carcinoma of the head and neck (HNSCC), and we have previously demonstrated that FA pathway depletion in HNSCC cell lines stimulates invasion. The goal of our studies was to use a systems approach in order to define FA pathway-dependent lipid metabolism and to extract lipid-based signatures and effectors of invasion in FA-deficient cells. Experimental Design: We subjected FA-isogenic HNSCC keratinocyte cell lines to untargeted and targeted lipidomics analyses to discover novel biomarkers and candidate therapeutic targets in FA-deficient cells. Cellular invasion assays were carried out in the presence and absence of N-butyldeoxynojirimycin (NB-DNJ), a biosynthetic inhibitor of the newly identified class of gangliosides, to investigate the requirement of ganglioside upregulation in FA-deficient HNSCC cells. Results: The most notable element of the lipid profiling results was a consistent elevation of glycosphingolipids, and particularly the accumulation of gangliosides. Conversely, repression of this same class of lipids was observed upon genetic correction of FA patient-derived HNSCC cells. Functional studies demonstrate that ganglioside upregulation is required for HNSCC cell invasion driven by FA pathway loss. The motility of nontransformed keratinocytes in response to FA loss displayed a similar dependence, thus supporting early and late roles for the FA pathway in controlling keratinocyte invasion through lipid regulation. Conclusions: Elevation of glycosphingolipids including the ganglioside GM3 in response to FA loss stimulates invasive characteristics of immortalized and transformed keratinocytes. An inhibitor of glycosphingolipid biosynthesis NB-DNJ attenuates invasive characteristics of FA-deficient HNSCC cells. Clin Cancer Res; 24(11); 2700-9. ©2018 AACR . ©2018 American Association for Cancer Research.

In Vivo Functional Selection Identifies Cardiotrophin-1 as a Cardiac Engraftment Factor for Mesenchymal Stromal Cells.

PubMed

Bortolotti, Francesca; Ruozi, Giulia; Falcione, Antonella; Doimo, Sara; Dal Ferro, Matteo; Lesizza, Pierluigi; Zentilin, Lorena; Banks, Lawrence; Zacchigna, Serena; Giacca, Mauro

2017-10-17

Transplantation of cells into the infarcted heart has significant potential to improve myocardial recovery; however, low efficacy of cell engraftment still limits therapeutic benefit. Here, we describe a method for the unbiased, in vivo selection of cytokines that improve mesenchymal stromal cell engraftment into the heart both in normal conditions and after myocardial infarction. An arrayed library of 80 secreted factors, including most of the currently known interleukins and chemokines, were individually cloned into adeno-associated viral vectors. Pools from this library were then used for the batch transduction of bone marrow-derived mesenchymal stromal cells ex vivo, followed by intramyocardial cell administration in normal and infarcted mice. Three weeks after injection, vector genomes were recovered from the few persisting cells and identified by sequencing DNA barcodes uniquely labeling each of the tested cytokines. The most effective molecule identified by this competitive engraftment screening was cardiotrophin-1, a member of the interleukin-6 family. Intracardiac injection of mesenchymal stromal cells transiently preconditioned with cardiotrophin-1 preserved cardiac function and reduced infarct size, parallel to the persistence of the transplanted cells in the healing hearts for at least 2 months after injection. Engraftment of cardiotrophin-1-treated mesenchymal stromal cells was consequent to signal transducer and activator of transcription 3-mediated activation of the focal adhesion kinase and its associated focal adhesion complex and the consequent acquisition of adhesive properties by the cells. These results support the feasibility of selecting molecules in vivo for their functional properties with adeno-associated viral vector libraries and identify cardiotrophin-1 as a powerful cytokine promoting cell engraftment and thus improving cell therapy of the infarcted myocardium. © 2017 American Heart Association, Inc.

Flight Test of the F/A-18 Active Aeroelastic Wing Airplane

NASA Technical Reports Server (NTRS)

Voracek, David

2007-01-01

A viewgraph presentation of flight tests performed on the F/A active aeroelastic wing airplane is shown. The topics include: 1) F/A-18 AAW Airplane; 2) F/A-18 AAW Control Surfaces; 3) Flight Test Background; 4) Roll Control Effectiveness Regions; 5) AAW Design Test Points; 6) AAW Phase I Test Maneuvers; 7) OBES Pitch Doublets; 8) OBES Roll Doublets; 9) AAW Aileron Flexibility; 10) Phase I - Lessons Learned; 11) Control Law Development and Verification & Validation Testing; 12) AAW Phase II RFCS Envelopes; 13) AAW 1-g Phase II Flight Test; 14) Region I - Subsonic 1-g Rolls; 15) Region I - Subsonic 1-g 360 Roll; 16) Region II - Supersonic 1-g Rolls; 17) Region II - Supersonic 1-g 360 Roll; 18) Region III - Subsonic 1-g Rolls; 19) Roll Axis HOS/LOS Comparison Region II - Supersonic (open-loop); 20) Roll Axis HOS/LOS Comparison Region II - Supersonic (closed-loop); 21) AAW Phase II Elevated-g Flight Test; 22) Region I - Subsonic 4-g RPO; and 23) Phase II - Lessons Learned

F/A-18 forebody vortex control. Volume 1: Static tests

NASA Technical Reports Server (NTRS)

Kramer, Brian R.; Suarez, Carlos J.; Malcolm, Gerald N.; Ayers, Bert F.

1994-01-01

A wind tunnel test was conducted on a six percent model of the F/A-18 at the NASA Ames 7 X 10-Foot Low Speed Wind Tunnel. The primary objective of the test was to evaluate several forebody vortex control configurations at high angles of attack in order to determine the most effective method of obtaining well behaved yawing moments, in preparation for the rotary balance test. Both mechanical and pneumatic systems were tested. Single and dual rotating nose tip strakes and a vertical nose strake were tested at different sizes and deflections. A series of jet blowing configurations were located at various fuselage stations, azimuth angles, and pointing angles ranging from straight aft to 60 deg canted inboard. Slot blowing was investigated for several slot lengths and fuselage stations. The effect of blowing rate was tested for both of these pneumatic systems. The most effective configurations were then further tested with a variation of both sideslip angle and Reynolds number over a range of angles of attack from 0 to 60 deg. It was found that a very robust system can be developed that provides yawing moments at angles of attack up to 60 deg that significantly exceeds that available from 30 deg of rudder deflection (F/A-18 maximum) at 0 deg angle of attack.

A Large-Scale RNAi Screen Identifies SGK1 as a Key Survival Kinase for GBM Stem Cells.

PubMed

Kulkarni, Shreya; Goel-Bhattacharya, Surbhi; Sengupta, Sejuti; Cochran, Brent H

2018-01-01

Glioblastoma multiforme (GBM) is the most common type of primary malignant brain cancer and has a very poor prognosis. A subpopulation of cells known as GBM stem-like cells (GBM-SC) have the capacity to initiate and sustain tumor growth and possess molecular characteristics similar to the parental tumor. GBM-SCs are known to be enriched in hypoxic niches and may contribute to therapeutic resistance. Therefore, to identify genetic determinants important for the proliferation and survival of GBM stem cells, an unbiased pooled shRNA screen of 10,000 genes was conducted under normoxic as well as hypoxic conditions. A number of essential genes were identified that are required for GBM-SC growth, under either or both oxygen conditions, in two different GBM-SC lines. Interestingly, only about a third of the essential genes were common to both cell lines. The oxygen environment significantly impacts the cellular genetic dependencies as 30% of the genes required under hypoxia were not required under normoxic conditions. In addition to identifying essential genes already implicated in GBM such as CDK4, KIF11 , and RAN , the screen also identified new genes that have not been previously implicated in GBM stem cell biology. The importance of the serum and glucocorticoid-regulated kinase 1 (SGK1) for cellular survival was validated in multiple patient-derived GBM stem cell lines using shRNA, CRISPR, and pharmacologic inhibitors. However, SGK1 depletion and inhibition has little effect on traditional serum grown glioma lines and on differentiated GBM-SCs indicating its specific importance in GBM stem cell survival. Implications: This study identifies genes required for the growth and survival of GBM stem cells under both normoxic and hypoxic conditions and finds SGK1 as a novel potential drug target for GBM. Mol Cancer Res; 16(1); 103-14. ©2017 AACR . ©2017 American Association for Cancer Research.

The cleaner, the greener? Product sustainability assessment of the biomimetic façade paint Lotusan® in comparison to the conventional façade paint Jumbosil®

PubMed Central

Antony, Florian; Grießhammer, Rainer; Speck, Thomas

2016-01-01

Background: The debate on the question whether biomimetics has a specific potential to contribute to sustainability is discussed among scientists, business leaders, politicians and those responsible for project funding. The objective of this paper is to contribute to this controversial debate by presenting the sustainability assessment of one of the most well-known and most successful biomimetic products: the façade paint Lotusan®. Results: As a first step it has been examined and verified that the façade paint Lotusan® is correctly defined as a biomimetic product. Secondly, Lotusan® has been assessed and compared to a conventional façade paint within the course of a detailed product sustainability assessment (PROSA). For purposes of comparison, the façade paint Jumbosil® was chosen as reference for a conventional paint available on the market. The benefit analysis showed that both paints fulfil equally well the requirements of functional utility. With respect to the symbolic utility, Lotusan® has a particular added aesthetic value by the preservation of the optical quality over the life cycle. Within the social analysis no substantial differences between the two paints could be found regarding the handling and disposal of the final products. Regarding the life-cycle cost, Lotusan® is the more expensive product. However, the higher investment cost for a Lotusan®-based façade painting are more than compensated by the longer life time, resulting in both reduced overall material demand and lower labour cost. In terms of the life-cycle impact assessment, it can be ascertained that substantial differences between the paints arise from the respective service life, which are presented in terms of four scenario analyses. Conclusion: In summary, the biomimetic façade paint Lotusan® has been identified as a cost-effective and at the same time resource-saving product. Based on the underlying data and assumptions it could be demonstrated that Lotusan®-based fa

The cleaner, the greener? Product sustainability assessment of the biomimetic façade paint Lotusan® in comparison to the conventional façade paint Jumbosil®.

PubMed

Antony, Florian; Grießhammer, Rainer; Speck, Thomas; Speck, Olga

2016-01-01

Background: The debate on the question whether biomimetics has a specific potential to contribute to sustainability is discussed among scientists, business leaders, politicians and those responsible for project funding. The objective of this paper is to contribute to this controversial debate by presenting the sustainability assessment of one of the most well-known and most successful biomimetic products: the façade paint Lotusan ® . Results: As a first step it has been examined and verified that the façade paint Lotusan ® is correctly defined as a biomimetic product. Secondly, Lotusan ® has been assessed and compared to a conventional façade paint within the course of a detailed product sustainability assessment (PROSA). For purposes of comparison, the façade paint Jumbosil ® was chosen as reference for a conventional paint available on the market. The benefit analysis showed that both paints fulfil equally well the requirements of functional utility. With respect to the symbolic utility, Lotusan ® has a particular added aesthetic value by the preservation of the optical quality over the life cycle. Within the social analysis no substantial differences between the two paints could be found regarding the handling and disposal of the final products. Regarding the life-cycle cost, Lotusan ® is the more expensive product. However, the higher investment cost for a Lotusan ® -based façade painting are more than compensated by the longer life time, resulting in both reduced overall material demand and lower labour cost. In terms of the life-cycle impact assessment, it can be ascertained that substantial differences between the paints arise from the respective service life, which are presented in terms of four scenario analyses. Conclusion: In summary, the biomimetic façade paint Lotusan ® has been identified as a cost-effective and at the same time resource-saving product. Based on the underlying data and assumptions it could be demonstrated that Lotusan �

Complementation of a Fanconi anemia group A cell line by UbA{sup 52}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moses, R.E.; Heina, J.A.; Jakobs, P.M.

1994-09-01

Cells from patients with Fanconi anemia (FA) display chromosomal instability and increased sensitivity to mitomycin C (MMC) and diepoxybutane (DEB) relative to normal cells. Several genes act in this pathway of DNA damage processing based upon four known complementation groups in FA. We have made a cDNA expression library in a vector with a G418 selectable marker to identify FA genes other than the FA-C group. Approximately 1 x 10{sup 6} independent cDNA clones were isolated with an average cDNA size of 1.5 kb. Five cell lines resistant to MMC and DEB were isolated from 6 x 10{sup 6} G418-resistantmore » transfectants from 65 individual transfections of the FA-A fibroblast line GM6914. The isolated cell lines also showed normal chromosome stability. The same cDNA (600 bp) was recovered from three independent cell lines by PCR using flanking sequence primers. The gene has sequence identity with a known gene, the ubiquitin fusion gene, UbA{sub 52}. Interestingly, each of the cDNAs were inserted in antisense orientation relative to the cytomegalovirus (CMV) promoter as determined by sequencing and PCR using UbA{sub 52}-specific internal primers. Southern blot analysis indicated the cell lines had distinct chromosomal insertion sites. Mutation analysis by chemical cleavage showed no reading frame mutations, indicating that UbA{sub 52} is not the FA-A gene. Re-transfection with the UbA{sub 52} gene in antisense gave complementation for MMC, DEB and chromosome stability to varying degrees. Re-transfection of the antisense construct with the CMV promotor removed or with a sense construct did not alter the MMC sensitivity. We conclude that the antisense UbA{sub 52} gene has a non-specific effect, perhaps acting by altering the cell cycle or susceptibility to apoptosis.« less

Synergistic inhibition of cancer cell proliferation with a combination of δ-tocotrienol and ferulic acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eitsuka, Takahiro, E-mail: eitsuka@nupals.ac.jp; Tatewaki, Naoto; Nishida, Hiroshi

2014-10-24

Highlights: • δ-Tocotrienol (δ-T3) and ferulic acid (FA) synergistically inhibit cancer cell growth. • The combination of δ-T3 and FA induces G1 arrest by up-regulating p21. • The synergy is attributed to an increase in the cellular concentration of δ-T3 by FA. - Abstract: Rice bran consists of many functional compounds and thus much attention has been focused on the health benefits of its components. Here, we investigated the synergistic inhibitory effects of its components, particularly δ-tocotrienol (δ-T3) and ferulic acid (FA), against the proliferation of an array of cancer cells, including DU-145 (prostate cancer), MCF-7 (breast cancer), and PANC-1more » (pancreatic cancer) cells. The combination of δ-T3 and FA markedly reduced cell proliferation relative to δ-T3 alone, and FA had no effect when used alone. Although δ-T3 induced G1 arrest by up-regulating p21 in PANC-1 cells, more cells accumulated in G1 phase with the combination of δ-T3 and FA. This synergistic effect was attributed to an increase in the cellular concentration of δ-T3 by FA. Our results suggest that the combination of δ-T3 and FA may present a new strategy for cancer prevention and therapy.« less

A Conserved Metal Binding Motif in the Bacillus subtilis Competence Protein ComFA Enhances Transformation.

PubMed

Chilton, Scott S; Falbel, Tanya G; Hromada, Susan; Burton, Briana M

2017-08-01

Genetic competence is a process in which cells are able to take up DNA from their environment, resulting in horizontal gene transfer, a major mechanism for generating diversity in bacteria. Many bacteria carry homologs of the central DNA uptake machinery that has been well characterized in Bacillus subtilis It has been postulated that the B. subtilis competence helicase ComFA belongs to the DEAD box family of helicases/translocases. Here, we made a series of mutants to analyze conserved amino acid motifs in several regions of B. subtilis ComFA. First, we confirmed that ComFA activity requires amino acid residues conserved among the DEAD box helicases, and second, we show that a zinc finger-like motif consisting of four cysteines is required for efficient transformation. Each cysteine in the motif is important, and mutation of at least two of the cysteines dramatically reduces transformation efficiency. Further, combining multiple cysteine mutations with the helicase mutations shows an additive phenotype. Our results suggest that the helicase and metal binding functions are two distinct activities important for ComFA function during transformation. IMPORTANCE ComFA is a highly conserved protein that has a role in DNA uptake during natural competence, a mechanism for horizontal gene transfer observed in many bacteria. Investigation of the details of the DNA uptake mechanism is important for understanding the ways in which bacteria gain new traits from their environment, such as drug resistance. To dissect the role of ComFA in the DNA uptake machinery, we introduced point mutations into several motifs in the protein sequence. We demonstrate that several amino acid motifs conserved among ComFA proteins are important for efficient transformation. This report is the first to demonstrate the functional requirement of an amino-terminal cysteine motif in ComFA. Copyright © 2017 American Society for Microbiology.

Production of Sophorolipid from an Identified Current Yeast, Lachancea thermotolerans BBMCZ7FA20, Isolated from Honey Bee.

PubMed

Mousavi, Fereshteh; Beheshti-Maal, Keivan; Massah, Ahmadreza

2015-08-01

Biosurfactants are a family of diverse amphipathic molecules that are produced by several microorganisms such as bacteria, molds, and yeasts. These surface active agents have several applications in agriculture, oil processing, food, and pharmaceutical industries. In this research using YMG and YUG culture media, a native yeast strain, HG5, was isolated from honey bee. The oil spread test as a screening method was used to evaluate biosurfactant production by the yeast HG5 isolate. The 5.8s-rDNA analysis confirmed that the isolated yeast was related to Lachancea thermotolerans. We named this strain Lachancea thermotolerans strain BBMCZ7FA20 and its 5.8s-rDNA sequence was deposited in GenBank, NCBI under accession number of KM042082.1. The best precursor of biosurfactant production was canola oil and the sophorolipid amount was measured for 24.2 g/l. The thin layer chromatography and Fourier Transform Infrared Spectroscopy analysis showed that the extracted biosurfactant from Lachancea thermotolerans was sophorolipid. In conclusion, this is the first report of sophorolipid production by a native yeast Lachancea thermotolerans BBMCZ7FA20 we isolated from the honey bee gut collected from an apiary farm in Saman, Chaharmahal Bakhtiari province, Iran. We suggested that some cost-effective supplements such as canola oil, sunflower oil, and corn oils could be applied for increasing the sophorolipid production by this native yeast strain. According to several applications of biosurfactants in today world, the production of sophorolipid by Lachancea thermotolerans could be considered as a potential in the current industrial microbiology and modern microbial biotechnology.

The RUNX1 +24 enhancer and P1 promoter identify a unique subpopulation of hematopoietic progenitor cells derived from human pluripotent stem cells

PubMed Central

Ferrell, Patrick I; Xi, Jiafei; Ma, Chao; Adlakha, Mitali; Kaufman, Dan S.

2016-01-01

Derivation of hematopoietic stem cells from human pluripotent stem cells remains a key goal for the fields of developmental biology and regenerative medicine. Here, we use a novel genetic reporter system to prospectively identify and isolate early hematopoietic cells derived from human embryonic stem cells (hESCs) and human induced pluripotent cells (iPSCs). Cloning the human RUNX1c P1 promoter and +24 enhancer to drive expression of tdTomato (tdTom) in hESCs and iPSCs, we demonstrate that tdTom expression faithfully enriches for RUNX1c-expressing hematopoietic progenitor cells. Time-lapse microscopy demonstrated the tdTom+ hematopoietic cells to emerge from adherent cells. Furthermore, inhibition of primitive hematopoiesis by blocking Activin/Nodal signaling promoted the expansion and/or survival of tdTom+ population. Notably, RUNX1c/tdTom+ cells represent only a limited subpopuation of CD34+CD45+ and CD34+CD43+ cells with a unique genetic signature. Using gene array analysis, we find significantly lower expression of Let-7 and mir181a microRNAs in the RUNX1c/tdTom+ cell population. These phenotypic and genetic analyses comparing the RUNX1c/tdTom+ population to CD34+CD45+ umbilical cord blood and fetal liver demonstrate several key differences that likely impact the development of HSCs capable of long-term multilineage engraftment from hESCs and iPSCs. PMID:25546363

Impaired TIP60-mediated H4K16 acetylation accounts for the aberrant chromatin accumulation of 53BP1 and RAP80 in Fanconi anemia pathway-deficient cells.

PubMed

Renaud, Emilie; Barascu, Aurelia; Rosselli, Filippo

2016-01-29

To rescue collapsed replication forks cells utilize homologous recombination (HR)-mediated mechanisms to avoid the induction of gross chromosomal abnormalities that would be generated by non-homologous end joining (NHEJ). Using DNA interstrand crosslinks as a replication barrier, we investigated how the Fanconi anemia (FA) pathway promotes HR at stalled replication forks. FA pathway inactivation results in Fanconi anemia, which is associated with a predisposition to cancer. FANCD2 monoubiquitination and assembly in subnuclear foci appear to be involved in TIP60 relocalization to the chromatin to acetylates histone H4K16 and prevents the binding of 53BP1 to its docking site, H4K20Me2. Thus, FA pathway loss-of-function results in accumulation of 53BP1, RIF1 and RAP80 at damaged chromatin, which impair DNA resection at stalled replication fork-associated DNA breaks and impede HR. Consequently, DNA repair in FA cells proceeds through the NHEJ pathway, which is likely responsible for the accumulation of chromosome abnormalities. We demonstrate that the inhibition of NHEJ or deacetylase activity rescue HR in FA cells. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Development and Characterization of a High Sensitivity Segmented Fast Neutron Spectrometer (FaNS-2)

PubMed Central

Langford, T.J.; Beise, E.J.; Breuer, H.; Heimbach, C.R.; Ji, G.; Nico, J.S.

2016-01-01

We present the development of a segmented fast neutron spectrometer (FaNS-2) based upon plastic scintillator and 3He proportional counters. It was designed to measure both the flux and spectrum of fast neutrons in the energy range of few MeV to 1 GeV. FaNS-2 utilizes capture-gated spectroscopy to identify neutron events and reject backgrounds. Neutrons deposit energy in the plastic scintillator before capturing on a 3He nucleus in the proportional counters. Segmentation improves neutron energy reconstruction while the large volume of scintillator increases sensitivity to low neutron fluxes. A main goal of its design is to study comparatively low neutron fluxes, such as cosmogenic neutrons at the Earth's surface, in an underground environment, or from low-activity neutron sources. In this paper, we present details of its design and construction as well as its characterization with a calibrated 252Cf source and monoenergetic neutron fields of 2.5 MeV and 14 MeV. Detected monoenergetic neutron spectra are unfolded using a Singular Value Decomposition method, demonstrating a 5% energy resolution at 14 MeV. Finally, we discuss plans for measuring the surface and underground cosmogenic neutron spectra with FaNS-2. PMID:27226807

Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified Clostridium botulinum Strain

PubMed Central

Pellett, Sabine; Tepp, William H.; Bradshaw, Marite; Kalb, Suzanne R.; Dykes, Janet K.; Lin, Guangyun; Nawrocki, Erin M.; Pier, Christina L.; Barr, John R.; Maslanka, Susan E.

2016-01-01

ABSTRACT Botulinum neurotoxins (BoNTs), produced by neurotoxigenic clostridial species, are the cause of the severe disease botulism in humans and animals. Early research on BoNTs has led to their classification into seven serotypes (serotypes A to G) based upon the selective neutralization of their toxicity in mice by homologous antibodies. Recently, a report of a potential eighth serotype of BoNT, designated “type H,” has been controversial. This novel BoNT was produced together with BoNT/B2 in a dual-toxin-producing Clostridium botulinum strain. The data used to designate this novel toxin as a new serotype were derived from culture supernatant containing both BoNT/B2 and novel toxin and from sequence information, although data from two independent laboratories indicated neutralization by antibodies raised against BoNT/A1, and classification as BoNT/FA was proposed. The sequence data indicate a chimeric structure consisting of a BoNT/A1 receptor binding domain, a BoNT/F5 light-chain domain, and a novel translocation domain most closely related to BoNT/F1. Here, we describe characterization of this toxin purified from the native strain in which expression of the second BoNT (BoNT/B) has been eliminated. Mass spectrometry analysis indicated that the toxin preparation contained only BoNT/FA and confirmed catalytic activity analogous to that of BoNT/F5. The in vivo mouse bioassay indicated a specific activity of this toxin of 3.8 × 107 mouse 50% lethal dose (mLD50) units/mg, whereas activity in cultured human neurons was very high (50% effective concentration [EC50] = 0.02 mLD50/well). Neutralization assays in cells and mice both indicated full neutralization by various antibodies raised against BoNT/A1, although at 16- to 20-fold-lower efficiency than for BoNT/A1. IMPORTANCE Botulinum neurotoxins (BoNTs), produced by anaerobic bacteria, are the cause of the potentially deadly, neuroparalytic disease botulism. BoNTs have been classified into seven serotypes

PW1 gene/paternally expressed gene 3 (PW1/Peg3) identifies multiple adult stem and progenitor cell populations

PubMed Central

Besson, Vanessa; Smeriglio, Piera; Wegener, Amélie; Relaix, Frédéric; Nait Oumesmar, Brahim; Sassoon, David A.; Marazzi, Giovanna

2011-01-01

A variety of markers are invaluable for identifying and purifying stem/progenitor cells. Here we report the generation of a murine reporter line driven by Pw1 that reveals cycling and quiescent progenitor/stem cells in all adult tissues thus far examined, including the intestine, blood, testis, central nervous system, bone, skeletal muscle, and skin. Neurospheres generated from the adult PW1-reporter mouse show near 100% reporter-gene expression following a single passage. Furthermore, epidermal stem cells can be purified solely on the basis of reporter-gene expression. These cells are clonogenic, repopulate the epidermal stem-cell niches, and give rise to new hair follicles. Finally, we demonstrate that only PW1 reporter-expressing epidermal cells give rise to follicles that are capable of self-renewal following injury. Our data demonstrate that PW1 serves as an invaluable marker for competent self-renewing stem cells in a wide array of adult tissues, and the PW1-reporter mouse serves as a tool for rapid stem cell isolation and characterization. PMID:21709251

Phenotypic correction of Fanconi anemia cells in the murine bone marrow after carrier cell mediated delivery of lentiviral vector.

PubMed

Chakkaramakkil Verghese, Santhosh; Goloviznina, Natalya A; Kurre, Peter

2016-11-19

Fanconi anemia (FA) is an autosomal-recessive disorder associated with hematopoietic failure and it is a candidate for hematopoietic stem cell (HSC)-directed gene therapy. However, the characteristically reduced HSC numbers found in FA patients, their ineffective mobilization from the marrow, and re-oxygenation damage during ex vivo manipulation have precluded clinical success using conventional in vitro approaches. We previously demonstrated that lentiviral vector (LV) particles reversibly attach to the cell surface where they gain protection from serum complement neutralization. We reasoned that cellular delivery of LV to the bone marrow niche could avoid detrimental losses during FA HSC mobilization and in vitro modification. Here, we demonstrate that a VSV-G pseudotyped lentivector, carrying the FANCC transgene, can be transmitted from carrier to bystander cells. In cell culture and transplantation models of FA, we further demonstrate that LV carrier cells migrate along SDF-1α gradients and transfer vector particles that stably integrate and phenotypically correct the characteristic DNA alkylator sensitivity in murine and human FA-deficient target bystander cells. Altogether, we demonstrate that cellular homing mechanisms can be harnessed for the functional phenotype correction in murine FA hematopoietic cells.

Folic acid inhibits COLO-205 colon cancer cell proliferation through activating the FRα/c-SRC/ERK1/2/NFκB/TP53 pathway: in vitro and in vivo studies

PubMed Central

Kuo, Chun-Ting; Chang, Chieh; Lee, Wen-Sen

2015-01-01

To investigate the molecular mechanism underlying folic acid (FA)-induced anti-colon caner activity, we showed that FA caused G0/G1 arrest in COLO-205. FA activated the proto-oncogene tyrosine-protein kinase Src (c-SRC)-mediated signaling pathway to enhance nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) nuclear translocation and binding onto the tumor protein p53 (TP53) gene promoter, and up-regulated expressions of TP53, cyclin-dependent kinase inhibitor 1A (CDKN1A) and cyclin-dependent kinase inhibitor 1B (CDKN1B). Knock-down of TP53 abolished FA-induced increases in the levels of CDKN1A and CDKN1B protein and G0/G1 arrest in COLO-205. Knock-down of folate receptor alpha (FRα) abolished FA-induced activations in the c-SRC-mediated pathway and increases in the levels of CDKN1A, CDKN1B and TP53 protein. These data suggest that FA inhibited COLO-205 proliferation through activating the FRα/c-SRC/mitogen-activated protein kinase 3/1 (ERK1/2)/NFκB/TP53 pathway-mediated up-regulations of CDKN1A and CDKN1B protein. In vivo studies demonstrated that daily i.p. injections of FA led to profound regression of the COLO-205 tumors and prolong the lifespan. In these tumors, the levels of CDKN1A, CDKN1B and TP53 protein were increased and von willebrand factor (VWF) protein levels were decreased. These findings suggest that FA inhibits COLO-205 colon cancer growth through anti-cancer cell proliferation and anti-angiogenesis. PMID:26056802

Volatile organic compounds produced by a soil-isolate, Bacillus subtilis FA26 induce adverse ultra-structural changes to the cells of Clavibacter michiganensis ssp. sepedonicus, the causal agent of bacterial ring rot of potato.

PubMed

Rajer, Faheem Uddin; Wu, Huijun; Xie, Yongli; Xie, Shanshan; Raza, Waseem; Tahir, Hafiz Abdul Samad; Gao, Xuewen

2017-04-01

Rhizobacterial volatile organic compounds (VOCs) play an important role in the suppression of soil-borne phytopathogens. In this study, the VOCs produced by a soil-isolate, Bacillus subtilis FA26, were evaluated in vitro for their antibacterial activity against Clavibacter michiganensis ssp. sepedonicus (Cms), the causal agent of bacterial ring rot of potato. The VOCs emitted by FA26 inhibited the growth of Cms significantly compared with the control. Scanning and transmission electron microscopy analyses revealed distorted colony morphology and a wide range of abnormalities in Cms cells exposed to the VOCs of FA26. Varying the inoculation strategy and inoculum size showed that the production and activity of the antibacterial VOCs of FA26 were dependent on the culture conditions. Headspace solid-phase microextraction/gas chromatography-mass spectrometry analyses revealed that FA26 produced 11 VOCs. Four VOCs (benzaldehyde, nonanal, benzothiazole and acetophenone) were associated with the antibacterial activity against Cms. The results suggested that the VOCs produced by FA26 could control the causal agent of bacterial ring rot of potato. This information will increase our understanding of the microbial interactions mediated by VOCs in nature and aid the development of safer strategies for controlling plant disease.

Detection of Gene Rearrangements in Circulating Tumor Cells: Examples of ALK-, ROS1-, RET-Rearrangements in Non-Small-Cell Lung Cancer and ERG-Rearrangements in Prostate Cancer.

PubMed

Catelain, Cyril; Pailler, Emma; Oulhen, Marianne; Faugeroux, Vincent; Pommier, Anne-Laure; Farace, Françoise

2017-01-01

Circulating tumor cells (CTCs) hold promise as biomarkers to aid in patient treatment stratification and disease monitoring. Because the number of cells is a critical parameter for exploiting CTCs for predictive biomarker's detection, we developed a FISH (fluorescent in situ hybridization) method for CTCs enriched on filters (filter-adapted FISH [FA-FISH]) that was optimized for high cell recovery. To increase the feasibility and reliability of the analyses, we combined fluorescent staining and FA-FISH and developed a semi-automated microscopy method for optimal FISH signal identification in filtration-enriched CTCs . Here we present these methods and their use for the detection and characterization of ALK-, ROS1-, RET-rearrangement in CTCs from non-small-cell lung cancer and ERG-rearrangements in CTCs from prostate cancer patients.

Metabolic Interaction between Anthocyanin and Lignin Biosynthesis Is Associated with Peroxidase FaPRX27 in Strawberry Fruit1[W

PubMed Central

Ring, Ludwig; Yeh, Su-Ying; Hücherig, Stephanie; Hoffmann, Thomas; Blanco-Portales, Rosario; Fouche, Mathieu; Villatoro, Carmen; Denoyes, Béatrice; Monfort, Amparo; Caballero, José Luis; Muñoz-Blanco, Juan; Gershenson, Jonathan; Schwab, Wilfried

2013-01-01

Plant phenolics have drawn increasing attention due to their potential nutritional benefits. Although the basic reactions of the phenolics biosynthetic pathways in plants have been intensively analyzed, the regulation of their accumulation and flux through the pathway is not that well established. The aim of this study was to use a strawberry (Fragaria × ananassa) microarray to investigate gene expression patterns associated with the accumulation of phenylpropanoids, flavonoids, and anthocyanins in strawberry fruit. An examination of the transcriptome, coupled with metabolite profiling data from different commercial varieties, was undertaken to identify genes whose expression correlated with altered phenolics composition. Seventeen comparative microarray analyses revealed 15 genes that were differentially (more than 200-fold) expressed in phenolics-rich versus phenolics-poor varieties. The results were validated by heterologous expression of the peroxidase FaPRX27 gene, which showed the highest altered expression level (more than 900-fold). The encoded protein was functionally characterized and is assumed to be involved in lignin formation during strawberry fruit ripening. Quantitative trait locus analysis indicated that the genomic region of FaPRX27 is associated with the fruit color trait. Down-regulation of the CHALCONE SYNTHASE gene and concomitant induction of FaPRX27 expression diverted the flux from anthocyanins to lignin. The results highlight the competition of the different phenolics pathways for their common precursors. The list of the 15 candidates provides new genes that are likely to impact polyphenol accumulation in strawberry fruit and could be used to develop molecular markers to select phenolics-rich germplasm. PMID:23835409

Birth order and avuncular tendencies in Samoan men and fa'afafine.

PubMed

Vanderlaan, Doug P; Vasey, Paul L

2013-04-01

Androphilia refers to sexual attraction and arousal to males whereas gynephilia refers to sexual attraction and arousal to females. In Samoa, transgendered androphilic males are known locally as fa'afafine. Previous research has shown that, compared to Samoan gynephilic men, fa'afafine report greater willingness to invest time and money toward nieces and nephews (i.e., greater avuncular tendencies) and also have greater numbers of older brothers and older sisters. The present study examined whether the Samoan male sexual orientation difference in avuncular tendencies could be accounted for by these parallel differences in numbers of older brothers and older sisters. The sample included 204 fa'afafine and 272 Samoan gynephilic men from our Samoan data archive for whom we had concurrent information on (1) a measure of willingness to invest time and money in nieces and nephews (i.e., avuncular tendencies) and (2) numbers of older and younger biological brothers and sisters. Among fa'afafine, but not Samoan gynephilic men, number of older brothers and number of older sisters were both significantly positively associated with avuncular tendencies. When controlling for number of older brothers, the magnitude of the male sexual orientation difference in avuncular tendencies was lowered, but remained statistically significant. In contrast, when controlling for number of older sisters, the male sexual orientation difference in avuncular tendencies ceased to exist. Discussion detailed how these findings help hone in on the proximate basis of elevated avuncular tendencies among fa'afafine. In addition, discussion focused on how particular evolutionary and cultural factors might relate to the avuncularity of fa'afafine.

Sca-1 Identifies a Distinct Androgen-Independent Murine Prostatic Luminal Cell Lineage with Bipotent Potential

PubMed Central

Kwon, Oh-Joon; Zhang, Li; Xin, Li

2016-01-01

Recent lineage tracing studies support the existence of prostate luminal progenitors that possess extensive regenerative capacity, but their identity remains unknown. We show that Sca-1 (Stem Cell Antigen-1) identifies a small population of murine prostate luminal cells that reside in the proximal prostatic ducts adjacent to the urethra. Sca-1+ luminal cells do not express Nkx3.1. They do not carry the secretory function, although they express the androgen receptor. These cells are enriched in the prostates of castrated mice. In the in vitro prostate organoid assay, a small fraction of the Sca-1+ luminal cells are capable of generating budding organoids that are morphologically distinct from those derived from other cell lineages. Histologically, this type of organoid is composed of multiple inner layers of luminal cells surrounded by multiple outer layers of basal cells. When passaged, these organoids retain their morphological and histological features. Finally, the Sca-1+ luminal cells are capable of forming small prostate glands containing both basal and luminal cells in an in vivo prostate regeneration assay. Collectively, our study establishes the androgen-independent and bipotent organoid-forming Sca-1+ luminal cells as a functionally distinct cellular entity. These cells may represent a putative luminal progenitor population and serve as a cellular origin for castration resistant prostate cancer. PMID:26418304

Utilization of flow cytometry to identify chimeral sectors in leaf tissue of Lolium multiflorum x L. arundinaceum hybrids

USDA-ARS?s Scientific Manuscript database

We have identified a method whereby Lolium multiflorum (Lm) or L. arundinaceum (Fa) genomes are preferentially eliminated through a mitotic loss behavior in interspecific Lm x Fa F1 hybrids, generating either dihaploid Lm lines or Fa lines. Flow cytometry, a method for rapidly characterizing optical...

Yamadazyma kitorensis f.a., sp. nov. and Zygoascus biomembranicola f.a., sp. nov., novel yeasts from the stone chamber interior of the Kitora tumulus, and five novel combinations in Yamadazyma and Zygoascus for species of Candida.

PubMed

Nagatsuka, Yuka; Ninomiya, Shinya; Kiyuna, Tomohiko; Kigawa, Rika; Sano, Chie; Sugiyama, Junta

2016-04-01

Analysis of D1/D2 large-subunit (LSU) rRNA gene sequences predicted that 17 yeast isolates, mainly from viscous gels (biofilms) taken from the stone chamber interior of the Kitora tumulus in Nara, Japan, were placed in the Yamadazyma and Zygoascus clades. Polyphasic characterization, including morphological, physiological and chemotaxonomic characteristics, multigene sequence divergence and DNA-DNA hybridization, strongly suggested the assignment of one novel species to each of the clades; these are Yamadazyma kitorensis f.a., sp. nov., with the type strain JCM 31005T (ex-type CBS 14158T=isolate K8617-6-8T), and Zygoascus biomembranicola f.a., sp. nov., with the type strain JCM 31007T (ex-type CBS 14157T=isolate K61208-2-11T). Furthermore, the transfer of five known species of the genus Candida as novel combinations to the genera Yamadazyma and Zygoascus is proposed; these are Yamadazyma olivae f.a., comb. nov. (type strain CBS 11171T=ATCC MYA-4568T), Yamadazyma tumulicola f.a., comb. nov. (type strain JCM 15403T=ex-type CBS 10917T=isolate T6517-9-5T), Yamadazyma takamatsuzukensis f.a., comb. nov. (type strain JCM 15410T=CBS 10916T = isolate T4922-1-1T), Zygoascus polysorbophila f.a., comb. nov. (type strain NRRL Y-27161T=CBS 7317T) and Zygoascus bituminiphila f.a., comb. nov. (type strain CBS 8813T=MUCL 41424T).

Aerodynamics of powered missile separation from F/A-18 aircraft

NASA Technical Reports Server (NTRS)

Ahmad, J. U.; Shanks, S. P.; Buning, P. G.

1993-01-01

A 3D dynamic 'chimera' algorithm that solves the thin-layer Navier-Stokes equations over multiple moving bodies was modified to numerically simulate the aerodynamics, missile dynamics, and missile plume interactions of a missile separating from a generic wing and from an F/A-18 aircraft in transonic flow. The missile is mounted below the wing for missile separation from the wing and on the F/A-18 fuselage at the engine inlet side for missile separation from aircraft. Static and powered missile separation cases are considered to examine the influence of the missile and plume on the wing and F/A-18 fuselage and engine inlet. The aircraft and missile are at two degrees angle of attack, Reynolds number of 10 million, freestream Mach number of 1.05 and plume Mach number of 3.0. The computational results show the details of the flow field.

Protein source in a high-protein diet modulates reductions in insulin resistance and hepatic steatosis in fa/fa Zucker rats.

PubMed

Wojcik, Jennifer L; Devassy, Jessay G; Wu, Yinghong; Zahradka, Peter; Taylor, Carla G; Aukema, Harold M

2016-01-01

High-protein diets are being promoted to reduce insulin resistance and hepatic steatosis in metabolic syndrome. Therefore, the effect of protein source in high-protein diets on reducing insulin resistance and hepatic steatosis was examined. Fa/fa Zucker rats were provided normal-protein (15% of energy) casein, high-protein (35% of energy) casein, high-protein soy, or high-protein mixed diets with animal and plant proteins. The high-protein mixed diet reduced area under the curve for insulin during glucose tolerance testing, fasting serum insulin and free fatty acid concentrations, homeostatic model assessment index, insulin to glucose ratio, and pancreatic islet cell area. The high-protein mixed and the high-protein soy diets reduced hepatic lipid concentrations, liver to body weight ratio, and hepatic steatosis rating. These improvements were observed despite no differences in body weight, feed intake, or adiposity among high-protein diet groups. The high-protein casein diet had minimal benefits. A high-protein mixed diet was the most effective for modulating reductions in insulin resistance and hepatic steatosis independent of weight loss, indicating that the source of protein within a high-protein diet is critical for the management of these metabolic syndrome parameters. © 2015 The Obesity Society.

Regulation of Chromatin Assembly and Cell Transformation by Formaldehyde Exposure in Human Cells

PubMed Central

Chen, Danqi; Fang, Lei; Mei, Shenglin; Li, Hongjie; Xu, Xia; Des Marais, Thomas L.; Lu, Kun; Liu, X. Shirley

2017-01-01

Background: Formaldehyde (FA) is an environmental and occupational chemical carcinogen. Recent studies have shown that exogenous FA causes only a modest increase in DNA adduct formation compared with the amount of adducts formed by endogenous FA, raising the possibility that epigenetic mechanisms may contribute to FA-mediated carcinogenicity. Objectives: We investigated the effects of FA exposure on histone modifications and chromatin assembly. We also examined the role of defective chromatin assembly in FA-mediated transcription and cell transformation. Methods: Cellular fractionation and Western blot analysis were used to measure the levels of histone modifications in human bronchial epithelial BEAS-2B cells and human nasal RPMI2650 cells in the presence of FA. Chromatin immunoprecipitation (ChIP) and micrococcal nuclease (MNase) digest assays were performed to examine the changes in chromatin assembly and accessibility after FA exposure. RNA sequencing (RNA-seq) and real-time polymerase chain reaction (PCR) were used to examine transcriptional dysregulation. Finally, anchorage-independent cell growth ability was tested by soft agar assay following FA exposure. Results: Exposure to FA dramatically decreased the acetylation of the N-terminal tails of cytosolic histones. These modifications are important for histone nuclear import and subsequent chromatin assembly. Histone proteins were depleted in both the chromatin fraction and at most of the genomic loci tested following FA exposure, suggesting that FA compromises chromatin assembly. Moreover, FA increased chromatin accessibility and altered the expression of hundreds of cancer-related genes. Knockdown of the histone H3.3 gene (an H3 variant), which mimics inhibition of chromatin assembly, facilitated FA-mediated anchorage-independent cell growth. Conclusions: We propose that the inhibition of chromatin assembly represents a novel mechanism of cell transformation induced by the environmental and occupational

Cortactin modulates RhoA activation and expression of Cip/Kip cyclin-dependent kinase inhibitors to promote cell cycle progression in 11q13-amplified head and neck squamous cell carcinoma cells.

PubMed

Croucher, David R; Rickwood, Danny; Tactacan, Carole M; Musgrove, Elizabeth A; Daly, Roger J

2010-11-01

The cortactin oncoprotein is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC), often due to amplification of the encoding gene (CTTN). While cortactin overexpression enhances invasive potential, recent research indicates that it also promotes cell proliferation, but how cortactin regulates the cell cycle machinery is unclear. In this article we report that stable short hairpin RNA-mediated cortactin knockdown in the 11q13-amplified cell line FaDu led to increased expression of the Cip/Kip cyclin-dependent kinase inhibitors (CDKIs) p21(WAF1/Cip1), p27(Kip1), and p57(Kip2) and inhibition of S-phase entry. These effects were associated with increased binding of p21(WAF1/Cip1) and p27(Kip1) to cyclin D1- and E1-containing complexes and decreased retinoblastoma protein phosphorylation. Cortactin regulated expression of p21(WAF1/Cip1) and p27(Kip1) at the transcriptional and posttranscriptional levels, respectively. The direct roles of p21(WAF1/Cip1), p27(Kip1), and p57(Kip2) downstream of cortactin were confirmed by the transient knockdown of each CDKI by specific small interfering RNAs, which led to partial rescue of cell cycle progression. Interestingly, FaDu cells with reduced cortactin levels also exhibited a significant diminution in RhoA expression and activity, together with decreased expression of Skp2, a critical component of the SCF ubiquitin ligase that targets p27(Kip1) and p57(Kip2) for degradation. Transient knockdown of RhoA in FaDu cells decreased expression of Skp2, enhanced the level of Cip/Kip CDKIs, and attenuated S-phase entry. These findings identify a novel mechanism for regulation of proliferation in 11q13-amplified HNSCC cells, in which overexpressed cortactin acts via RhoA to decrease expression of Cip/Kip CDKIs, and highlight Skp2 as a downstream effector for RhoA in this process.

NEW CHEMICAL ALTERNATIVE FOR OZONE-DEPLETING SUBSTANCES: HFC-236FA

EPA Science Inventory

The report gives results of a preliminary evaluation of a new hydrofluorocarbon (HFC)--HFC-236fa or 1,1,1,3,3,3-hexafluoropropane--as a possible alternative for chlorofluorocarbon (CFC)-114 (1,2-dichloro-1,1,2,2-tetrafluoroethane) refrigerant for chillers and as a possible fire s...

Fanconi anemia genes are highly expressed in primitive CD34+ hematopoietic cells

PubMed Central

Aubé, Michel; Lafrance, Matthieu; Brodeur, Isabelle; Delisle, Marie-Chantal; Carreau, Madeleine

2003-01-01

Background Fanconi anemia (FA) is a complex recessive genetic disease characterized by progressive bone marrow failure (BM) and a predisposition to cancer. We have previously shown using the Fancc mouse model that the progressive BM failure results from a hematopoietic stem cell defect suggesting that function of the FA genes may reside in primitive hematopoietic stem cells. Methods Since genes involved in stem cell differentiation and/or maintenance are usually regulated at the transcription level, we used a semiquantitative RT-PCR method to evaluate FA gene transcript levels in purified hematopoietic stem cells. Results We show that most FA genes are highly expressed in primitive CD34-positive and negative cells compared to lower levels in more differentiated cells. However, in CD34- stem cells the Fancc gene was found to be expressed at low levels while Fancg was undetectable in this population. Furthermore, Fancg expression is significantly decreased in Fancc -/- stem cells as compared to wild-type cells while the cancer susceptibility genes Brca1 and Fancd1/Brac2 are upregulated in Fancc-/- hematopoietic cells. Conclusions These results suggest that FA genes are regulated at the mRNA level, that increased Fancc expression in LTS-CD34+ cells correlates with a role at the CD34+ differentiation stage and that lack of Fancc affects the expression of other FA gene, more specifically Fancg and Fancd1/Brca2, through an unknown mechanism. PMID:12809565

Orientations of Iron-Sulfur Clusters FA and FB in the Homodimeric Type-I Photosynthetic Reaction Center of Heliobacterium modesticaldum.

PubMed

Kondo, Toru; Matsuoka, Masahiro; Azai, Chihiro; Itoh, Shigeru; Oh-Oka, Hirozo

2016-05-12

Orientations of the FA and FB iron-sulfur (FeS) clusters in a structure-unknown type-I homodimeric heriobacterial reaction center (hRC) were studied in oriented membranes of the thermophilic anaerobic photosynthetic bacterium Heliobacterium modesticaldum by electron paramagnetic resonance (EPR), and compared with those in heterodimeric photosystem I (PS I). The Rieske-type FeS center in the cytochrome b/c complex showed a well-oriented EPR signal. Illumination at 14 K induced an FB(-) signal with g-axes of gz = 2.066, gy = 1.937, and gx = 1.890, tilted at angles of 60°, 60°, and 45°, respectively, with respect to the membrane normal. Chemical reduction with dithionite produced an additional signal of FA(-), which magnetically interacted with FB(-), with gz = 2.046, gy = 1.942, and gx = 1.911 at 30°, 60°, and 90°, respectively. The angles and redox properties of FA(-) and FB(-) in hRC resemble those of FB(-) and FA(-), respectively, in PS I. Therefore, FA and FB in hRC, named after their g-value similarities, seem to be located like FB and FA, not like FA and FB, respectively, in PS I. The reducing side of hRC could resemble those in PS I, if the names of FA and FB are interchanged with each other.

F/A-18 1/9th scale model tail buffet measurements

NASA Technical Reports Server (NTRS)

Martin, C. A.; Glaister, M. K.; Maclaren, L. D.; Meyn, L. A.; Ross, J.

1991-01-01

Wind tunnel tests were carried out on a 1/9th scale model of the F/A-18 at high angles of attack to investigate the characteristics of tail buffet due to bursting of the wing leading edge extension (LEX) vortices. The tests were carried out at the Aeronautical Research Laboratory low-speed wind tunnel facility and form part of a collaborative activity with NASA Ames Research Center, organized by The Technical Cooperative Program (TTCP). Information from the program will be used in the planning of similar collaborative tests, to be carried out at NASA Ames, on a full-scale aircraft. The program covered the measurement of unsteady pressures and fin vibration for cases with and without the wing LEX fences fitted. Fourier transform methods were used to analyze the unsteady data, and information on the spatial and temporal content of the vortex burst pressure field was obtained. Flow visualization of the vortex behavior was carried out using smoke and a laser light sheet technique.

Functional Analysis of Problem Behavior: A Systematic Approach for Identifying Idiosyncratic Variables

PubMed Central

Roscoe, Eileen M.; Schlichenmeyer, Kevin J.; Dube, William V.

2015-01-01

When inconclusive functional analysis (FA) outcomes occur, a number of modifications have been made to enhance the putative establishing operation or consequence associated with behavioral maintenance. However, a systematic method for identifying relevant events to test during modified FAs has not been evaluated. The purpose of this study was to develop and evaluate a technology for systematically identifying events to test in a modified FA after an initial FA led to inconclusive outcomes. Six individuals whose initial FA showed little or no responding or high levels only in the control condition participated. An indirect assessment (IA) questionnaire developed for identifying idiosyncratic variables was administered, and a descriptive analysis (DA) was conducted. Results from the IA only or a combination of the IA and DA were used to inform modified FA test and control conditions. Conclusive FA outcomes were obtained with five of the six participants during the modified FA phase. PMID:25930176

Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells

NASA Astrophysics Data System (ADS)

Lu, Shaoying; Seong, Jihye; Wang, Yi; Chang, Shiou-Chi; Eichorst, John Paul; Ouyang, Mingxing; Li, Julie Y.-S.; Chien, Shu; Wang, Yingxiao

2014-07-01

Focal adhesions (FAs) are dynamic subcellular structures crucial for cell adhesion, migration and differentiation. It remains an enigma how enzymatic activities in these local complexes regulate their structural remodeling in live cells. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity, although cells displayed heterogeneous levels of response to stimulation. Within lipid rafts, the time delay between Src activation and FA disassembly was 1.2 min in cells seeded on low fibronectin concentration ([FN]) and 4.3 min in cells on high [FN]. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions, as a tight enzyme-structure coupling occurred in FA populations mediated by integrin αvβ3, but not in those by integrin α5β1. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics.

FaRXf1: a locus conferring resistance to angular leaf spot caused by Xanthomonas fragariae in octoploid strawberry.

PubMed

Roach, Jack A; Verma, Sujeet; Peres, Natalia A; Jamieson, Andrew R; van de Weg, W Eric; Bink, Marco C A M; Bassil, Nahla V; Lee, Seonghee; Whitaker, Vance M

2016-06-01

Angular leaf spot is a devastating bacterial disease of strawberry. Resistance from two wild accessions is highly heritable and controlled by a major locus on linkage group 6D. Angular leaf spot caused by Xanthomonas fragariae is the only major bacterial disease of cultivated strawberry (Fragaria ×ananassa). While this disease may cause reductions of up to 8 % of marketable yield in Florida winter annual production, no resistant cultivars have been commercialized. Wild accessions US4808 and US4809 were previously identified as resistant to the four genetic clades of X. fragariae, and introgression of the trait into commercial quality perennial-type germplasm was initiated. Previous reports indicated high heritability for the trait but proposed both single-locus and multi-locus inheritance models. The objective of this study was to determine the mode of inheritance of resistance, to identify causal loci, and to begin introgression of resistance into Florida-adapted germplasm. Resistance was observed in two years of field trials with inoculated plants that assayed four full-sib families descended from US4808 to US4809. Resistance segregated 1:1 in all families indicating control by a dominant allele at a single locus. Using a selective genotyping approach with the IStraw90 Axiom(®) SNP array and pedigree-based QTL detection, a single major-effect QTL was identified in two full-sib families, one descended from each resistant accession. High-resolution melt curve analysis validated the presence of the QTL in separate populations. The QTL was delimited to the 33.1-33.6 Mbp (F. vesca vesca v1.1 reference) and 34.8-35.3 Mbp (F. vesca bracteata v2.0 reference) regions of linkage group 6D for both resistance sources and was designated FaRXf1. Characterization of this locus will facilitate marker-assisted selection toward the development of resistant cultivars.

FA-SAT Is an Old Satellite DNA Frozen in Several Bilateria Genomes

PubMed Central

Chaves, Raquel; Ferreira, Daniela; Mendes-da-Silva, Ana; Meles, Susana; Adega, Filomena

2017-01-01

Abstract In recent years, a growing body of evidence has recognized the tandem repeat sequences, and specifically satellite DNA, as a functional class of sequences in the genomic “dark matter.” Using an original, complementary, and thus an eclectic experimental design, we show that the cat archetypal satellite DNA sequence, FA-SAT, is “frozen” conservatively in several Bilateria genomes. We found different genomic FA-SAT architectures, and the interspersion pattern was conserved. In Carnivora genomes, the FA-SAT-related sequences are also amplified, with the predominance of a specific FA-SAT variant, at the heterochromatic regions. We inspected the cat genome project to locate FA-SAT array flanking regions and revealed an intensive intermingling with transposable elements. Our results also show that FA-SAT-related sequences are transcribed and that the most abundant FA-SAT variant is not always the most transcribed. We thus conclude that the DNA sequences of FA-SAT and their transcripts are “frozen” in these genomes. Future work is needed to disclose any putative function that these sequences may play in these genomes. PMID:29608678

TRIM.FaTE Evaluation Report

EPA Pesticide Factsheets

The TRIM.FaTE Evaluation Report is composed of three volumes. Volume I presents conceptual, mechanistic, and structural complexity evaluations of various aspects of the model. Volumes II and III present performance evaluation.

Integrated genomic analyses identify WEE1 as a critical mediator of cell fate and novel therapeutic target in acute myeloid leukemia

PubMed Central

Porter, Christopher C.; Kim, Jihye; Fosmire, Susan; Gearheart, Christy M.; van Linden, Annemie; Baturin, Dmitry; Zaberezhnyy, Vadym; Patel, Purvi R.; Gao, Dexiang; Tan, Aik Choon; DeGregori, James

2011-01-01

Acute myeloid leukemia (AML) remains a therapeutic challenge despite increasing knowledge about the molecular origins of the disease, as the mechanisms of AML cell escape from chemotherapy remain poorly defined. We hypothesized that AML cells are addicted to molecular pathways in the context of chemotherapy and used complementary approaches to identify these addictions. Using novel molecular and computational approaches, we performed genome-wide shRNA screens to identify proteins that mediate AML cell fate after cytarabine exposure, gene expression profiling of AML cells exposed to cytarabine to identify genes with induced expression in this context, and examination of existing gene expression data from primary patient samples. The integration of these independent analyses strongly implicates cell cycle checkpoint proteins, particularly WEE1, as critical mediators of AML cell survival after cytarabine exposure. Knockdown of WEE1 in a secondary screen confirmed its role in AML cell survival. Pharmacologic inhibition of WEE1 in AML cell lines and primary cells is synergistic with cytarabine. Further experiments demonstrate that inhibition of WEE1 prevents S-phase arrest induced by cytarabine, broadening the functions of WEE1 that may be exploited therapeutically. These data highlight the power of integrating functional and descriptive genomics, and identify WEE1 as potential therapeutic target in AML. PMID:22289989

Microarray identifies ADAM family members as key responders to TGF-beta1 in alveolar epithelial cells.

PubMed

Keating, Dominic T; Sadlier, Denise M; Patricelli, Andrea; Smith, Sinead M; Walls, Dermot; Egan, Jim J; Doran, Peter P

2006-09-01

The molecular mechanisms of Idiopathic Pulmonary Fibrosis (IPF) remain elusive. Transforming Growth Factor beta 1(TGF-beta1) is a key effector cytokine in the development of lung fibrosis. We used microarray and computational biology strategies to identify genes whose expression is significantly altered in alveolar epithelial cells (A549) in response to TGF-beta1, IL-4 and IL-13 and Epstein Barr virus. A549 cells were exposed to 10 ng/ml TGF-beta1, IL-4 and IL-13 at serial time points. Total RNA was used for hybridisation to Affymetrix Human Genome U133A microarrays. Each in vitro time-point was studied in duplicate and an average RMA value computed. Expression data for each time point was compared to control and a signal log ratio of 0.6 or greater taken to identify significant differential regulation. Using normalised RMA values and unsupervised Average Linkage Hierarchical Cluster Analysis, a list of 312 extracellular matrix (ECM) proteins or modulators of matrix turnover was curated via Onto-Compare and Gene-Ontology (GO) databases for baited cluster analysis of ECM associated genes. Interrogation of the dataset using ontological classification focused cluster analysis revealed coordinate differential expression of a large cohort of extracellular matrix associated genes. Of this grouping members of the ADAM (A disintegrin and Metalloproteinase domain containing) family of genes were differentially expressed. ADAM gene expression was also identified in EBV infected A549 cells as well as IL-13 and IL-4 stimulated cells. We probed pathologenomic activities (activation and functional activity) of ADAM19 and ADAMTS9 using siRNA and collagen assays. Knockdown of these genes resulted in diminished production of collagen in A549 cells exposed to TGF-beta1, suggesting a potential role for these molecules in ECM accumulation in IPF.

Starmerella reginensis f.a., sp. nov. and Starmerella kourouensis f.a., sp. nov., isolated from flowers in French Guiana.

PubMed

Amoikon, Tiemele Laurent Simon; Grondin, Cécile; Djéni, Théodore N'Dédé; Jacques, Noémie; Casaregola, Serge

2018-05-21

Analysis of yeasts isolated from various biotopes in French Guiana led to the identification of two strains isolated from flowers and designated CLIB 1634 T and CLIB 1707 T . Comparison of the D1/D2 domain of the large subunit (LSU D1/D2) rRNA gene sequences of CLIB 1634 T and CLIB 1707 T to those in the GenBank database revealed that these strains belong to the Starmerella clade. Strain CLIB 1634 T was shown to diverge from the closely related Starmerella apicola type strain CBS 2868 T with a sequence divergence of 1.34 and 1.30 %, in the LSU D1/D2 rRNA gene and internal transcribed spacer (ITS) sequences respectively. Strain CLIB 1634 T and Candida apicola CBS 2868 T diverged by 3.81 and 14.96 % at the level of the protein-coding gene partial sequences EF-1α and RPB2, respectively. CLIB 1707 T was found to have sequence divergence of 3.88 and 9.16 % in the LSU D1/D2 rRNA gene and ITS, respectively, from that of the most closely related species Starmerella ratchasimensis type strain CBS 10611 T . The species Starmerella reginensis f.a., sp. nov. and Starmerella kourouensis f.a., sp. nov. are proposed to accommodate strains CLIB 1634 T (=CBS 15247 T ) and CLIB 1707 T (=CBS 15257 T ), respectively.

Targeted disruption of exons 1 to 6 of the Fanconi Anemia group A gene leads to growth retardation, strain-specific microphthalmia, meiotic defects and primordial germ cell hypoplasia.

PubMed

Wong, Jasmine C Y; Alon, Noa; Mckerlie, Colin; Huang, Jun R; Meyn, M Stephen; Buchwald, Manuel

2003-08-15

Fanconi Anemia (FA) is an autosomal recessive disorder characterized by cellular hypersensitivity to DNA cross-linking agents. Recent studies suggest that FA proteins share a common pathway with BRCA proteins. To study the in vivo role of the FA group A gene (Fanca), gene-targeting techniques were used to generate Fanca(tm1Hsc) mice in which Fanca exons 1-6 were replaced by a beta-galactosidase reporter construct. Fanca(tm1.1Hsc) mice were generated by Cre-mediated removal of the neomycin cassette in Fanca(tm1Hsc) mice. Fanca(tm1.1Hsc) homozygotes display FA-like phenotypes including growth retardation, microphthalmia and craniofacial malformations that are not found in other Fanca mouse models, and the genetic background affects manifestation of certain phenotypes. Both male and female mice homozygous for Fanca mutation exhibit hypogonadism, and homozygous females demonstrate premature reproductive senescence and an increased incidence of ovarian cysts. We showed that fertility defects in Fanca(tm1.1Hsc) homozygotes might be related to a diminished population of primordial germ cells (PGCs) during migration into the gonadal ridges. We also found a high level of Fanca expression in pachytene spermatocytes. Fanca(tm1Hsc) homozygous males exhibited an elevated frequency of mispaired meiotic chromosomes and increased apoptosis in germ cells, implicating a role for Fanca in meiotic recombination. However, the localization of Rad51, Brca1, Fancd2 and Mlh1 appeared normal on Fanca(tm1Hsc) homozygous meiotic chromosomes. Taken together, our results suggest that the FA pathway plays a role in the maintenance of reproductive germ cells and in meiotic recombination.

Weekly infusional high-dose fluorouracil (HD-FU), HD-FU plus folinic acid (HD-FU/FA), or HD-FU/FA plus biweekly cisplatin in advanced gastric cancer: randomized phase II trial 40953 of the European Organisation for Research and Treatment of Cancer Gastrointestinal Group and the Arbeitsgemeinschaft Internistische Onkologie.

PubMed

Lutz, Manfred P; Wilke, Hansjochen; Wagener, D J Theo; Vanhoefer, Udo; Jeziorski, Krzysztof; Hegewisch-Becker, Susanna; Balleisen, Leopold; Joossens, Eric; Jansen, Rob L; Debois, Muriel; Bethe, Ullrich; Praet, Michel; Wils, Jacques; Van Cutsem, Eric

2007-06-20

This multicentric, randomized, two-stage phase II trial evaluated three simplified weekly infusional regimens of fluorouracil (FU) or FU plus folinic acid (FA) and cisplatin (Cis) with the aim to select a regimen for future phase III trials. A total of 145 patients with advanced gastric cancer where randomly assigned to weekly FU 3,000 mg/m2/24 hours (HD-FU), FU 2,600 mg/m2/24 hours plus dl-FA 500 mg/m2 or l-FA 250 mg/m2 (HD-FU/FA), or FU 2000 mg/m2/24 hours plus FA plus biweekly Cis 50 mg/m2, each administered for 6 weeks with a 1-week rest. The primary end point was the response rate. Confirmed responses were observed in 6.1% (two of 33) of the eligible patients treated with HD-FU, in 25% (12 of 48, including one complete remission [CR]) with HD-FU/FA, and in 45.7% (21 of 46, including four CRs) with HD-FU/FA/Cis. The HD-FU arm was closed after stage 1 because the required minimum number of responses was not met. The median progression-free survival of all patients in the HD-FU, HD-FU/FA, and HD-FU/FA/Cis arm was 1.9, 4.0, and 6.1 months, respectively. The median overall survival was 7.1, 8.9, and 9.7 months, and the survival rate at 1 year was 24.3%, 30.3%, and 45.3%, respectively. Grade 4 toxicities were rare. The most relevant grade 3/4 toxicities were neutropenia in 1.9%, 5.4%, and 19.6%, and diarrhea in 2.7%, 1.9%, and 3.9% of the cycles in the HD-FU, HD-FU/FA, and HD-/FU/Cis arms, respectively. Weekly infusional FU/FA plus biweekly Cis is effective and safe in patients with gastric cancer.

STAT3-activated CD36 facilitates fatty acid uptake in chronic lymphocytic leukemia cells

PubMed Central

Rozovski, Uri; Harris, David M.; Li, Ping; Liu, Zhiming; Jain, Preetesh; Ferrajoli, Alessandra; Burger, Jan; Thompson, Phillip; Jain, Nitin; Wierda, William; Keating, Michael J.; Estrov, Zeev

2018-01-01

Although several studies established that unlike normal B cells chronic lymphocytic leukemia (CLL) cells metabolize fatty acids (FA), how CLL cells internalize FA is poorly understood. Because in various cell types CD36 facilitates FA uptake, we wondered whether a similar mechanism is operative CLL. We found that CD36 levels are higher in CLL cells than in normal B cells, and that small interfering RNA, CD36 neutralizing antibodies or sulfosuccinimidyl oleate (SSO) that inhibits CD36 significantly reduced the oxygen consumption of CLL cells incubated with FA. Because CD36 is oeverexpressed and STAT3 is constitutively activated in CLL cells, we wondered whether STAT3 induces CD36 expression. Sequence analysis identified putative STAT3 binding sites in the CD36 gene promoter. Chromatin immunoprecipitation and an electrophoretic mobility shift assay revealed that STAT3 binds to the CD36 gene promoter. A luciferase assay and STAT3-small hairpin RNA, that significantly decreased the levels of CD36 in CLL cells, established that STAT3 activates the transcription of the CD36 gene. Furthermore, SSO induced a dose-dependent apoptosis of CLL cells. Taken together, our data suggest that STAT3 activates CD36 and that CD36 facilitates FA uptake in CLL cells. Whether CD36 inhibition would provide clinical benefits in CLL remains to be determined. PMID:29765537

Adaptive Façade: Variant-Finding using Shape Grammar

NASA Astrophysics Data System (ADS)

Tomasowa, Riva; Utama Sjarifudin, Firza

2017-12-01

Modular façade construction has never been better since the birth of computer-aided manufacturing which bridges the modeling phase into the manufacturing phase for escalating the mass production. This comes to a result that the identity of a product or a building façade will commonly generate in the same way that the initial design was intended to. Rectifying the early model will then greatly impact the process later. The aim of this paper is to propose a way to solve these two challenges, without risking the manufacturing process, but more to explore the potential designs. Shape grammar is used to conceive more designs in the early stage, derived from the initial product - the modular adaptive façade system. The derivations are then tested through simulation to state the efficacy of the models. We find that the workflow somehow contributes to the better design and engineering process as well as the solution allows diversification in the façade expressions.

Effect of Folic Acid and Vitamin B12 on Pemetrexed Antifolate Chemotherapy in Nutrient Lung Cancer Cells

PubMed Central

Yang, Tsung-Ying; Chang, Gee-Chen; Hsu, Shih-Lan; Huang, Yi-Rou; Chiu, Ling-Yen

2013-01-01

Pemetrexed (MTA) is a multitargeted antifolate drug approved for lung cancer therapy. Clinically, supplementation with high doses of folic acid (FA) and vitamin B12 (VB12) lowers MTA cytotoxicities. An antagonistic effect of FA/VB12 on MTA efficacy has been proposed. However, patients who receive FA/VB12 show better tolerance to MTA with improved survival. The aims of this study are to investigate the modulation of FA and VB12 on MTA drug efficacy in human nonsmall cell lung cancer (NSCLC) cell lines. The sensitivities of cells, apoptosis, and MTA-regulated proteins were characterized to determine the possible effects of high doses of FA and VB12 on MTA efficacy. MTA has the lowest efficacy under 10% serum conditions. However, supplementation with FA and VB12 individually and additively reversed the insensitivity of NSCLC cells to MTA treatment with 10% serum. The enhanced sensitivities of cells following FA/VB12 treatment were correlated with increasing apoptosis and were specific to MTA but not to 5-fluorouracil (5-FU). Enhanced sensitivity was also associated with p21WAF1/Cip1 expression level. Our results revealed no antagonistic effect of high doses of FA/VB12 on MTA efficacy in cancer cells grown in nutrient medium. Furthermore, these data may partially explain why supplementation of FA and VB12 resulted in better survival in MTA-treated patients. PMID:23984356

FaStore - a space-saving solution for raw sequencing data.

PubMed

Roguski, Lukasz; Ochoa, Idoia; Hernaez, Mikel; Deorowicz, Sebastian

2018-03-29

The affordability of DNA sequencing has led to the generation of unprecedented volumes of raw sequencing data. These data must be stored, processed, and transmitted, which poses significant challenges. To facilitate this effort, we introduce FaStore, a specialized compressor for FASTQ files. FaStore does not use any reference sequences for compression, and permits the user to choose from several lossy modes to improve the overall compression ratio, depending on the specific needs. FaStore in the lossless mode achieves a significant improvement in compression ratio with respect to previously proposed algorithms. We perform an analysis on the effect that the different lossy modes have on variant calling, the most widely used application for clinical decision making, especially important in the era of precision medicine. We show that lossy compression can offer significant compression gains, while preserving the essential genomic information and without affecting the variant calling performance. FaStore can be downloaded from https://github.com/refresh-bio/FaStore. sebastian.deorowicz@polsl.pl. Supplementary data are available at Bioinformatics online.

Hydrogen peroxide production and mitochondrial dysfunction contribute to the fusaric acid-induced programmed cell death in tobacco cells.

PubMed

Jiao, Jiao; Sun, Ling; Zhou, Benguo; Gao, Zhengliang; Hao, Yu; Zhu, Xiaoping; Liang, Yuancun

2014-08-15

Fusaric acid (FA), a non-specific toxin produced mainly by Fusarium spp., can cause programmed cell death (PCD) in tobacco suspension cells. The mechanism underlying the FA-induced PCD was not well understood. In this study, we analyzed the roles of hydrogen peroxide (H2O2) and mitochondrial function in the FA-induced PCD. Tobacco suspension cells were treated with 100 μM FA and then analyzed for H2O2 accumulation and mitochondrial functions. Here we demonstrate that cells undergoing FA-induced PCD exhibited H2O2 production, lipid peroxidation, and a decrease of the catalase and ascorbate peroxidase activities. Pre-treatment of tobacco suspension cells with antioxidant ascorbic acid and NADPH oxidase inhibitor diphenyl iodonium significantly reduced the rate of FA-induced cell death as well as the caspase-3-like protease activity. Moreover, FA treatment of tobacco cells decreased the mitochondrial membrane potential and ATP content. Oligomycin and cyclosporine A, inhibitors of the mitochondrial ATP synthase and the mitochondrial permeability transition pore, respectively, could also reduce the rate of FA-induced cell death significantly. Taken together, the results presented in this paper demonstrate that H2O2 accumulation and mitochondrial dysfunction are the crucial events during the FA-induced PCD in tobacco suspension cells. Copyright © 2014 Elsevier GmbH. All rights reserved.

Enhanced anticancer activity of DM1-loaded star-shaped folate-core PLA-TPGS nanoparticles

NASA Astrophysics Data System (ADS)

Tang, Xiaolong; Liang, Yong; Zhu, Yongqiang; Cai, Shiyu; Sun, Leilei; Chen, Tianyi

2014-10-01

The efficient delivery of therapeutic drugs into interested cells is a critical challenge to broad application of nonviral vector systems. In this research, emtansine (DM1)-loaded star-shaped folate-core polylactide- d-α-tocopheryl polyethylene glycol 1000 succinate (FA-PLA-TPGS-DM1) copolymer which demonstrated superior anticancer activity in vitro/ vivo in comparison with linear FA-PLA-TPGS nanoparticles was applied to be a vector of DM1 for FR+ breast cancer therapy. The DM1- or coumarin 6-loaded nanoparticles were fabricated, and then characterized in terms of size, morphology, drug encapsulation efficiency, and in vitro drug release. And the viability of MCF-7/HER2 cells treated with FA-DM1-nanoparticles (NPs) was assessed. Severe combined immunodeficient mice carrying MCF-7/HER2 tumor xenografts were treated in several groups including phosphate-buffered saline control, DM1, DM1-NPs, and FA-DM1-NPs. The antitumor activity was then assessed by survival time and solid tumor volume. All the specimens were prepared for formalin-fixed and paraffin-embedded tissue sections for hematoxylin-eosin staining. The data showed that the FA-DM1-NPs could efficiently deliver DM1 into MCF-7/HER2 cells. The cytotoxicity of DM1 to MCF-7/HER2 cells was significantly increased by FA-DM1-NPs when compared with the control groups. In conclusion, the FA-DM1-NPs offered a considerable potential formulation for FR+ tumor-targeting biotherapy.

Metabolic engineering of E. coli top 10 for production of vanillin through FA catabolic pathway and bioprocess optimization using RSM.

PubMed

Chakraborty, Debkumar; Gupta, Gaganjot; Kaur, Baljinder

2016-12-01

Metabolic engineering and construction of recombinant Escherichia coli strains carrying feruloyl-CoA synthetase and enoyl-CoA hydratase genes for the bioconversion of ferulic acid to vanillin offers an alternative way to produce vanillin. Isolation and designing of fcs and ech genes was carried out using computer assisted protocol and the designed vanillin biosynthetic gene cassette was cloned in pCCIBAC expression vector for introduction in E. coli top 10. Recombinant strain was implemented for the statistical optimization of process parameters influencing F A to vanillin biotransformation. CCD matrix constituted of process variables like FA concentration, time, temperature and biomass with intracellular, extracellular and total vanillin productions as responses. Production was scaled up and 68 mg/L of vanillin was recovered from 10 mg/L of FA using cell extracts from 1 mg biomass within 30 min. Kinetic activity of enzymes were characterized. From LCMS-ESI analysis a metabolic pathway of FA degradation and vanillin production was predicted. Copyright © 2016 Elsevier Inc. All rights reserved.

Gene expression profile in circulating mononuclear cells after exposure to ultrafine carbon particles

PubMed Central

Huang, Yuh-Chin T.; Schmitt, Michael; Yang, Zhonghui; Que, Loretta G.; Stewart, Judith C.; Frampton, Mark W.; Devlin, Robert B.

2013-01-01

Context Exposure to particulate matter (PM) is associated with systemic health effects, but the cellular and molecular mechanisms are unclear. Objective We hypothesized that, if circulating mononuclear cells play an important role in mediating systemic effects of PM, they would show gene expression changes following exposure. Materials and methods Peripheral blood samples were collected before (0 hour) and at 24 hours after exposure from healthy subjects who participated in previous controlled exposures to ultrafine carbon particles (UFP, 50 μg/m3) or filtered air (FA)(n = 3 each). RNA from mononuclear cell fraction (>85% lymphocytes) was extracted, amplified and hybridized to Affymetrix HU133 plus 2 microarrays. Results We identified 1713 genes (UFP 24 hours vs. FA 0 and 24 hours, p < 0.05, FDR 0.01). The top 10 upregulated genes (fold) were CDKN1C (1.86), ZNF12 (1.83), SRGAP2 (1.82), FYB (1.79), LSM14B (1.79), CD93 (1.76), NCSTN (1.70), DUSP6 (1.69), TACC1 (1.68) and H2AFY (1.68). Upregulation of CDKN1C and SRGAP2 was confirmed by RT-PCR using samples from additional 5 subjects exposed to FA and UFP. We entered 1020 genes with a ratio >1.1 or <−1.1 into the Ingenuity Pathway Analysis and identified many canonical pathways related to inflammation, tissue growth and host defense against environmental insults, including IGF-1 signaling, insulin receptor signaling and NRF2-mediated oxidative stress response pathway. Discussion and conclusions Two-hour exposures to UFP produced gene expression changes in circulating mononuclear cells. These gene changes provide biologically plausible links to PM-induced systemic health effects, especially those in the cardiovascular system and glucose metabolism. PMID:20507211

Proteome analysis identifies L1CAM/CD171 and DPP4/CD26 as novel markers of human skin mast cells.

PubMed

Gschwandtner, M; Paulitschke, V; Mildner, M; Brunner, P M; Hacker, S; Eisenwort, G; Sperr, W R; Valent, P; Gerner, C; Tschachler, E

2017-01-01

The function of skin mast cells has been well documented in IgE-mediated allergic reactions, whereas other mast cell functions are poorly defined. This study aimed at identifying novel mast cell proteins by proteome analysis of primary human skin mast cells. The proteome of skin mast cells was compared to other cell types and analyzed using bioinformatics. The expression and function of two proteins hitherto not described in skin mast cells was investigated in isolated mast cells as well as in mast cells in situ. Within the mast cell proteome, we identified 49 highly expressed proteins previously not described in mast cells; 21 of these proteins were found to be selectively expressed in mast cells. Two proteins, the neural cell adhesion molecule L1 and dipeptidyl peptidase 4, were further studied. L1 was found to be highly expressed in mast cells in normal, psoriasis, and mastocytosis skin. Dipeptidyl peptidase 4 was found to be expressed in mast cells in normal, psoriasis, and mastocytosis skin as well as in bone marrow mast cells in patients with systemic mastocytosis. In normal skin, mast cells were identified as a major source of dipeptidyl peptidase 4 and we also found that skin mast cells and fibroblasts secrete an active form of this enzyme. In a systematic proteomics approach we identified two novel mast cell proteins potentially relevant to skin homeostasis: neural cell adhesion molecule L1 and dipeptidyl peptidase 4. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Fluorophore Absorption Size Exclusion Chromatography (FA-SEC): An Alternative Method for High-Throughput Detergent Screening of Membrane Proteins.

PubMed

Lin, Sung-Yao; Sun, Xing-Han; Hsiao, Yu-Hsuan; Chang, Shao-En; Li, Guan-Syun; Hu, Nien-Jen

2016-01-01

Membrane proteins play key roles in many fundamental functions in cells including ATP synthesis, ion and molecule transporter, cell signalling and enzymatic reactions, accounting for ~30% genes of whole genomes. However, the hydrophobic nature of membrane proteins frequently hampers the progress of structure determination. Detergent screening is the critical step in obtaining stable detergent-solubilized membrane proteins and well-diffracting protein crystals. Fluorescence Detection Size Exclusion Chromatography (FSEC) has been developed to monitor the extraction efficiency and monodispersity of membrane proteins in detergent micelles. By tracing the FSEC profiles of GFP-fused membrane proteins, this method significantly enhances the throughput of detergent screening. However, current methods to acquire FSEC profiles require either an in-line fluorescence detector with the SEC equipment or an off-line spectrofluorometer microplate reader. Here, we introduce an alternative method detecting the absorption of GFP (FA-SEC) at 485 nm, thus making this methodology possible on conventional SEC equipment through the in-line absorbance spectrometer. The results demonstrate that absorption is in great correlation with fluorescence of GFP. The comparably weaker absorption signal can be improved by using a longer path-length flow cell. The FA-SEC profiles were congruent with the ones plotted by FSEC, suggesting FA-SEC could be a comparable and economical setup for detergent screening of membrane proteins.

Proteasome activity is important for replication recovery, CHK1 phosphorylation and prevention of G2 arrest after low-dose formaldehyde

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ortega-Atienza, Sara; Green, Samantha E.; Zhitkovich, Anatoly, E-mail: anatoly_zhitkovich@brown.edu

2015-07-15

Formaldehyde (FA) is a human carcinogen with numerous sources of environmental and occupational exposures. This reactive aldehyde is also produced endogenously during metabolism of drugs and other processes. DNA–protein crosslinks (DPCs) are considered to be the main genotoxic lesions for FA. Accumulating evidence suggests that DPC repair in high eukaryotes involves proteolysis of crosslinked proteins. Here, we examined a role of the main cellular proteolytic machinery proteasomes in toxic responses of human lung cells to low FA doses. We found that transient inhibition of proteasome activity increased cytotoxicity and diminished clonogenic viability of FA-treated cells. Proteasome inactivation exacerbated suppressive effectsmore » of FA on DNA replication and increased the levels of the genotoxic stress marker γ-H2AX in normal human cells. A transient loss of proteasome activity in FA-exposed cells also caused delayed perturbations of cell cycle, which included G2 arrest and a depletion of S-phase populations at FA doses that had no effects in control cells. Proteasome activity diminished p53-Ser15 phosphorylation but was important for FA-induced CHK1 phosphorylation, which is a biochemical marker of DPC proteolysis in replicating cells. Unlike FA, proteasome inhibition had no effect on cell survival and CHK1 phosphorylation by the non-DPC replication stressor hydroxyurea. Overall, we obtained evidence for the importance of proteasomes in protection of human cells against biologically relevant doses of FA. Biochemically, our findings indicate the involvement of proteasomes in proteolytic repair of DPC, which removes replication blockage by these highly bulky lesions. - Highlights: • Proteasome inhibition enhances cytotoxicity of low-dose FA in human lung cells. • Active proteasomes diminish replication-inhibiting effects of FA. • Proteasome activity prevents delayed G2 arrest in FA-treated cells. • Proteasome inhibition exacerbates replication stress

miRNomes of haematopoietic stem cells and dendritic cells identify miR-30b as a regulator of Notch1

PubMed Central

Su, Xiaoping; Qian, Cheng; Zhang, Qian; Hou, Jin; Gu, Yan; Han, Yanmei; Chen, Yongjian; Jiang, Minghong; Cao, Xuetao

2013-01-01

Dendritic cells (DCs) are critical to initiate the immune response and maintain tolerance, depending on different status and subsets. The expression profiles of microRNAs (miRNAs) in various DC subsets and haematopoietic stem cells (HSCs), which generate DCs, remain to be fully identified. Here we examine miRNomes of mouse bone marrow HSCs, immature DCs, mature DCs and IL-10/NO-producing regulatory DCs by deep sequencing. We identify numerous stage-specific miRNAs and histone modification in HSCs and DCs at different differentiation stages. miR-30b, significantly upregulated via a TGF-beta/Smad3-mediated epigenetic pathway in regulatory DCs, can target Notch1 to promote IL-10 and NO production, suggesting that miR-30b is a negative regulator of immune response. We also identify miRNomes of in vivo counterparts of mature DCs and regulatory DCs and systematically compare them with DCs cultured in vitro. These results provide a resource for studying roles of miRNAs in stem cell biology, development and functional regulation of DC subsets. PMID:24309499

The inhibition of c-MYC transcription factor modulates the expression of glycolytic and glutaminolytic enzymes in FaDu hypopharyngeal carcinoma cells.

PubMed

Kleszcz, Robert; Paluszczak, Jarosław; Krajka-Kuźniak, Violetta; Baer-Dubowska, Wanda

2018-05-17

Cancer cells are dependent on aerobic glycolysis for energy production and increased glutamine consumption. HIF-1α and c-MYC transcription factors regulate the expression of glycolytic and glutaminolytic genes. Their activity may be repressed by SIRT6. Head and neck carcinomas show frequent activation of c-MYC function and SIRT6 down-regulation, which contributes to a strong dependence on glucose and glutamine availability. The aim of this study was to compare the influence of HIF-1α and c-MYC inhibitors (KG-548 and 10058-F4, respectively) and potential SIRT6 inducers - resveratrol and its synthetic derivative DMU-212 with the effect of glycolysis and glutaminolysis inhibitors (2-deoxyglucose and aminooxyacetic acid, respectively) on the metabolism and expression of metabolic enzymes in FaDu hypopharyngeal carcinoma cells. Cell viability was assessed by means of an MTT assay. Quantitative PCR was performed to evaluate the expression of SIRT6, HIF-1α, c-MYC, GLUT1, SLC1A5, HK2, PFKM, PKM2, LDHA, GLS, and GDH. The release of glycolysis and glutaminolysis end-products into the culture medium - lactate and ammonia, respectively - was assessed using standard colorimetric assays. Lactate production was significantly inhibited by 10058-F4, KG-548, and 2-deoxyglucose. Moreover, 10058-F4 strongly reduced the amount of ammonia release. The effects of 10058-F4 activity can be attributed to a reduction in the expression of PKM2 and LDHA. On the other hand, the induction of SIRT6 expression by resveratrol and DMU-212 was not associated with significant modulation of the expression of metabolic enzymes. Overall, the results of this study indicate that the inhibition of c-MYC may be considered to be a promising strategy of the modulation of cancer-related metabolic changes in head and neck carcinomas.

Fatty Acid Biosynthesis Inhibition Increases Reduction Potential in Neuronal Cells under Hypoxia.

PubMed

Brose, Stephen A; Golovko, Svetlana A; Golovko, Mikhail Y

2016-01-01

Recently, we have reported a novel neuronal specific pathway for adaptation to hypoxia through increased fatty acid (FA) biosynthesis followed by esterification into lipids. However, the biological role of this pathway under hypoxia remains to be elucidated. In the presented study, we have tested our hypothesis that activation of FA synthesis maintains reduction potential and reduces lactoacidosis in neuronal cells under hypoxia. To address this hypothesis, we measured the effect of FA synthesis inhibition on [Formula: see text]/NAD + and [Formula: see text]/NADP + ratios, and lactic acid levels in neuronal SH-SY5Y cells exposed to normoxic and hypoxic conditions. FA synthesis inhibitors, TOFA (inhibits Acetyl-CoA carboxylase) and cerulenin (inhibits FA synthase), increased [Formula: see text]/NAD + and [Formula: see text]/NADP + ratios under hypoxia. Further, FA synthesis inhibition increased lactic acid under both normoxic and hypoxic conditions, and caused cytotoxicity under hypoxia but not normoxia. These results indicate that FA may serve as hydrogen acceptors under hypoxia, thus supporting oxidation reactions including anaerobic glycolysis. These findings may help to identify a radically different approach to attenuate hypoxia related pathophysiology in the nervous system including stroke.

Fatty Acid Biosynthesis Inhibition Increases Reduction Potential in Neuronal Cells under Hypoxia

PubMed Central

Brose, Stephen A.; Golovko, Svetlana A.; Golovko, Mikhail Y.

2016-01-01

Recently, we have reported a novel neuronal specific pathway for adaptation to hypoxia through increased fatty acid (FA) biosynthesis followed by esterification into lipids. However, the biological role of this pathway under hypoxia remains to be elucidated. In the presented study, we have tested our hypothesis that activation of FA synthesis maintains reduction potential and reduces lactoacidosis in neuronal cells under hypoxia. To address this hypothesis, we measured the effect of FA synthesis inhibition on NADH2+/NAD+ and NADPH2+/NADP+ ratios, and lactic acid levels in neuronal SH-SY5Y cells exposed to normoxic and hypoxic conditions. FA synthesis inhibitors, TOFA (inhibits Acetyl-CoA carboxylase) and cerulenin (inhibits FA synthase), increased NADH2+/NAD+ and NADPH2+/NADP+ ratios under hypoxia. Further, FA synthesis inhibition increased lactic acid under both normoxic and hypoxic conditions, and caused cytotoxicity under hypoxia but not normoxia. These results indicate that FA may serve as hydrogen acceptors under hypoxia, thus supporting oxidation reactions including anaerobic glycolysis. These findings may help to identify a radically different approach to attenuate hypoxia related pathophysiology in the nervous system including stroke. PMID:27965531

White Bass (Morone chrysops) Preferentially Retain n-3 PUFA in Ova When Fed Prepared Diets with Varying FA Content.

PubMed

Fuller, S Adam; Rawles, Steven D; McEntire, Matthew E; Bader, Troy J; Riche, Marty; Beck, Benjamin H; Webster, Carl D

2017-10-01

We evaluated the fatty acid (FA) composition of broodstock white bass ova fed one of six commercial diets with increasing polyunsaturated FA content (n-6/n-3 ratio; 0.36, 0.39, 0.46, 0.83, 1.07, 1.12) eight weeks prior to sampling. Fatty acid profiles of ova from brooders fed each of the six diets were significantly altered according to canonical discriminant analysis. Ova FA profiles resulting from the 0.39 diet separated those from the 0.36 diet based on lower 18:2n-6 (LNA) and higher 20:1n-9 concentrations from the 0.36 diet. Ova profiles were further separated based on lower concentrations of 22:5n-3 (DPA) from the 0.46 diet, lower concentrations of 20:5n-3 (EPA) in the 1.12 and 0.83 diets, and lower concentrations of 22:6n-3 (DHA) in all other diets relative to the 0.46 diet. Changes in ova FA profile at four and eight weeks were consistent with dietary intake with an approximate 2% increase in any given FA class with increasing time on individual diet. There was no correlation between dietary ARA concentrations (0.7-1.1 mol%), or dietary EPA/ARA ratios (7-15), and the concentrations (1.4-1.7 mol%) or ratios (3.3-4.4) found in the ova by diet. Our results suggest that white bass females have the ability to preferentially incorporate n-3 PUFA, particularly DHA, suggesting mobilization of this FA from other tissues for ova deposition or preferential dietary incorporation of PUFA into ova. These results will add to the limited FA information available in white bass and enable nutritionists to formulate broodstock diets that maximize reproductive potential in this species.

The Pattern of Fatty Acids Displaced by EPA and DHA Following 12 Months Supplementation Varies between Blood Cell and Plasma Fractions.

PubMed

Walker, Celia G; West, Annette L; Browning, Lucy M; Madden, Jackie; Gambell, Joanna M; Jebb, Susan A; Calder, Philip C

2015-08-03

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are increased in plasma lipids and blood cell membranes in response to supplementation. Whilst arachidonic acid (AA) is correspondingly decreased, the effect on other fatty acids (FA) is less well described and there may be site-specific differences. In response to 12 months EPA + DHA supplementation in doses equivalent to 0-4 portions of oily fish/week (1 portion: 3.27 g EPA+DHA) multinomial regression analysis was used to identify important FA changes for plasma phosphatidylcholine (PC), cholesteryl ester (CE) and triglyceride (TAG) and for blood mononuclear cells (MNC), red blood cells (RBC) and platelets (PLAT). Dose-dependent increases in EPA + DHA were matched by decreases in several n-6 polyunsaturated fatty acids (PUFA) in PC, CE, RBC and PLAT, but were predominantly compensated for by oleic acid in TAG. Changes were observed for all FA classes in MNC. Consequently the n-6:n-3 PUFA ratio was reduced in a dose-dependent manner in all pools after 12 months (37%-64% of placebo in the four portions group). We conclude that the profile of the FA decreased in exchange for the increase in EPA + DHA following supplementation differs by FA pool with implications for understanding the impact of n-3 PUFA on blood lipid and blood cell biology.

The Pattern of Fatty Acids Displaced by EPA and DHA Following 12 Months Supplementation Varies between Blood Cell and Plasma Fractions

PubMed Central

Walker, Celia G.; West, Annette L.; Browning, Lucy M.; Madden, Jackie; Gambell, Joanna M.; Jebb, Susan A.; Calder, Philip C.

2015-01-01

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are increased in plasma lipids and blood cell membranes in response to supplementation. Whilst arachidonic acid (AA) is correspondingly decreased, the effect on other fatty acids (FA) is less well described and there may be site-specific differences. In response to 12 months EPA + DHA supplementation in doses equivalent to 0–4 portions of oily fish/week (1 portion: 3.27 g EPA+DHA) multinomial regression analysis was used to identify important FA changes for plasma phosphatidylcholine (PC), cholesteryl ester (CE) and triglyceride (TAG) and for blood mononuclear cells (MNC), red blood cells (RBC) and platelets (PLAT). Dose-dependent increases in EPA + DHA were matched by decreases in several n-6 polyunsaturated fatty acids (PUFA) in PC, CE, RBC and PLAT, but were predominantly compensated for by oleic acid in TAG. Changes were observed for all FA classes in MNC. Consequently the n-6:n-3 PUFA ratio was reduced in a dose-dependent manner in all pools after 12 months (37%–64% of placebo in the four portions group). We conclude that the profile of the FA decreased in exchange for the increase in EPA + DHA following supplementation differs by FA pool with implications for understanding the impact of n-3 PUFA on blood lipid and blood cell biology. PMID:26247960

Overcoming reprogramming resistance of Fanconi anemia cells

PubMed Central

Müller, Lars U. W.; Milsom, Michael D.; Harris, Chad E.; Vyas, Rutesh; Brumme, Kristina M.; Parmar, Kalindi; Moreau, Lisa A.; Schambach, Axel; Park, In-Hyun; London, Wendy B.; Strait, Kelly; Schlaeger, Thorsten; DeVine, Alexander L.; Grassman, Elke; D'Andrea, Alan; Daley, George Q.

2012-01-01

Fanconi anemia (FA) is a recessive syndrome characterized by progressive fatal BM failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway that is critical for maintaining genomic integrity and protecting cells from the DNA damage caused by cross-linking agents. Transgenic expression of the implicated genes corrects the phenotype of hematopoietic cells, but previous attempts at gene therapy have failed largely because of inadequate numbers of hematopoietic stem cells available for gene correction. Induced pluripotent stem cells (iPSCs) constitute an alternate source of autologous cells that are amenable to ex vivo expansion, genetic correction, and molecular characterization. In the present study, we demonstrate that reprogramming leads to activation of the FA pathway, increased DNA double-strand breaks, and senescence. We also demonstrate that defects in the FA DNA-repair pathway decrease the reprogramming efficiency of murine and human primary cells. FA pathway complementation reduces senescence and restores the reprogramming efficiency of somatic FA cells to normal levels. Disease-specific iPSCs derived in this fashion maintain a normal karyotype and are capable of hematopoietic differentiation. These data define the role of the FA pathway in reprogramming and provide a strategy for future translational applications of patient-specific FA iPSCs. PMID:22371882

Control Law-Control Allocation Interaction: F/A-18 PA Simulation Test - Bed

NASA Technical Reports Server (NTRS)

Durham, Wayne; Nelson, Mark

2001-01-01

This report documents the first stage of research into Control Law - Control Allocation Interactions. A three-year research effort was originally proposed: 1. Create a desktop flight simulation environment under which experiments related to the open questions may be conducted. 2. Conduct research to determine which aspects of control allocation have impact upon control law design that merits further research. 3. Conduct research into those aspects of control allocation identified above, and their impacts upon control law design. Simulation code was written utilizing the F/A-18 airframe in the power approach (PA) configuration. A dynamic inversion control law was implemented and used to drive a state-of-the-art control allocation subroutine.

A Targeted RNAi Screen Identifies Endocytic Trafficking Factors That Control GLP-1 Receptor Signaling in Pancreatic β-Cells.

PubMed

Buenaventura, Teresa; Kanda, Nisha; Douzenis, Phoebe C; Jones, Ben; Bloom, Stephen R; Chabosseau, Pauline; Corrêa, Ivan R; Bosco, Domenico; Piemonti, Lorenzo; Marchetti, Piero; Johnson, Paul R; Shapiro, A M James; Rutter, Guy A; Tomas, Alejandra

2018-03-01

The glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) is a key target for type 2 diabetes (T2D) treatment. Because endocytic trafficking of agonist-bound receptors is one of the most important routes for regulation of receptor signaling, a better understanding of this process may facilitate the development of new T2D therapeutic strategies. Here, we screened 29 proteins with known functions in G protein-coupled receptor trafficking for their role in GLP-1R potentiation of insulin secretion in pancreatic β-cells. We identify five (clathrin, dynamin1, AP2, sorting nexins [SNX] SNX27, and SNX1) that increase and four (huntingtin-interacting protein 1 [HIP1], HIP14, GASP-1, and Nedd4) that decrease insulin secretion from murine insulinoma MIN6B1 cells in response to the GLP-1 analog exendin-4. The roles of HIP1 and the endosomal SNX1 and SNX27 were further characterized in mouse and human β-cell lines and human islets. While HIP1 was required for the coupling of cell surface GLP-1R activation with clathrin-dependent endocytosis, the SNXs were found to control the balance between GLP-1R plasma membrane recycling and lysosomal degradation and, in doing so, determine the overall β-cell incretin responses. We thus identify key modulators of GLP-1R trafficking and signaling that might provide novel targets to enhance insulin secretion in T2D. © 2017 by the American Diabetes Association.

In silico approaches to identify novel myeloid cell leukemia-1 (Mcl-1) inhibitors for treatment of cancer.

PubMed

Ren, Ji-Xia; Li, Cheng-Ping; Zhou, Xiu-Ling; Cao, Xue-Song; Xie, Yong

2017-08-22

Myeloid cell leukemia-1 (Mcl-1) has been a validated and attractive target for cancer therapy. Over-expression of Mcl-1 in many cancers allows cancer cells to evade apoptosis and contributes to the resistance to current chemotherapeutics. Here, we identified new Mcl-1 inhibitors using a multi-step virtual screening approach. First, based on two different ligand-receptor complexes, 20 pharmacophore models were established by simultaneously using 'Receptor-Ligand Pharmacophore Generation' method and manual build feature method, and then carefully validated by a test database. Then, pharmacophore-based virtual screening (PB-VS) could be performed by using the 20 pharmacophore models. In addition, docking study was used to predict the possible binding poses of compounds, and the docking parameters were optimized before performing docking-based virtual screening (DB-VS). Moreover, a 3D QSAR model was established by applying the 55 aligned Mcl-1 inhibitors. The 55 inhibitors sharing the same scaffold were docked into the Mcl-1 active site before alignment, then the inhibitors with possible binding conformations were aligned. For the training set, the 3D QSAR model gave a correlation coefficient r 2 of 0.996; for the test set, the correlation coefficient r 2 was 0.812. Therefore, the developed 3D QSAR model was a good model, which could be applied for carrying out 3D QSAR-based virtual screening (QSARD-VS). After the above three virtual screening methods orderly filtering, 23 potential inhibitors with novel scaffolds were identified. Furthermore, we have discussed in detail the mapping results of two potent compounds onto pharmacophore models, 3D QSAR model, and the interactions between the compounds and active site residues.

Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis

PubMed Central

Mizuno, Takako; Sridharan, Anusha; Du, Yina; Guo, Minzhe; Wikenheiser-Brokamp, Kathryn A.; Perl, Anne-Karina T.; Funari, Vincent A.; Gokey, Jason J.; Stripp, Barry R.; Whitsett, Jeffrey A.

2016-01-01

Idiopathic pulmonary fibrosis (IPF) is a lethal interstitial lung disease characterized by airway remodeling, inflammation, alveolar destruction, and fibrosis. We utilized single-cell RNA sequencing (scRNA-seq) to identify epithelial cell types and associated biological processes involved in the pathogenesis of IPF. Transcriptomic analysis of normal human lung epithelial cells defined gene expression patterns associated with highly differentiated alveolar type 2 (AT2) cells, indicated by enrichment of RNAs critical for surfactant homeostasis. In contrast, scRNA-seq of IPF cells identified 3 distinct subsets of epithelial cell types with characteristics of conducting airway basal and goblet cells and an additional atypical transitional cell that contributes to pathological processes in IPF. Individual IPF cells frequently coexpressed alveolar type 1 (AT1), AT2, and conducting airway selective markers, demonstrating “indeterminate” states of differentiation not seen in normal lung development. Pathway analysis predicted aberrant activation of canonical signaling via TGF-β, HIPPO/YAP, P53, WNT, and AKT/PI3K. Immunofluorescence confocal microscopy identified the disruption of alveolar structure and loss of the normal proximal-peripheral differentiation of pulmonary epithelial cells. scRNA-seq analyses identified loss of normal epithelial cell identities and unique contributions of epithelial cells to the pathogenesis of IPF. The present study provides a rich data source to further explore lung health and disease. PMID:27942595

Solvent-Free Addition of Indole to Aldehydes: Unexpected Synthesis of Novel 1-[1-(1H-Indol-3-yl) Alkyl]-1H-Indoles and Preliminary Evaluation of Their Cytotoxicity in Hepatocarcinoma Cells.

PubMed

Tocco, Graziella; Zedda, Gloria; Casu, Mariano; Simbula, Gabriella; Begala, Michela

2017-10-17

New 1-[1-(1 H -indol-3-yl) alkyl]-1 H -indoles, surprisingly, have been obtained from the addition of indole to a variety of aldehydes under neat conditions. CaO, present in excess, was fundamental for carrying out the reaction with paraformaldehyde. Under the same reaction conditions, aromatic and heteroaromatic aldehydes afforded only classical bis (indolyl) aryl indoles. In this paper, the role of CaO, together with the regiochemistry and the mechanism of the reaction, are discussed in detail. The effect of some selected 3,3'- and 1,3'-diindolyl methane derivatives on cell proliferation of the hepatoma cell line FaO was also evaluated.

Apn1 AP-endonuclease is essential for the repair of oxidatively damaged DNA bases in yeast frataxin-deficient cells.

PubMed

Lefevre, Sophie; Brossas, Caroline; Auchère, Françoise; Boggetto, Nicole; Camadro, Jean-Michel; Santos, Renata

2012-09-15

Frataxin deficiency results in mitochondrial dysfunction and oxidative stress and it is the cause of the hereditary neurodegenerative disease Friedreich ataxia (FA). Here, we present evidence that one of the pleiotropic effects of oxidative stress in frataxin-deficient yeast cells (Δyfh1 mutant) is damage to nuclear DNA and that repair requires the Apn1 AP-endonuclease of the base excision repair pathway. Major phenotypes of Δyfh1 cells are respiratory deficit, disturbed iron homeostasis and sensitivity to oxidants. These phenotypes are weak or absent under anaerobiosis. We show here that exposure of anaerobically grown Δyfh1 cells to oxygen leads to down-regulation of antioxidant defenses, increase in reactive oxygen species, delay in G1- and S-phases of the cell cycle and damage to mitochondrial and nuclear DNA. Nuclear DNA lesions in Δyfh1 cells are primarily caused by oxidized bases and single-strand breaks that can be detected 15-30 min after oxygen exposition. The Apn1 enzyme is essential for the repair of the DNA lesions in Δyfh1 cells. Compared with Δyfh1, the double Δyfh1Δapn1 mutant shows growth impairment, increased mutagenesis and extreme sensitivity to H(2)O(2). On the contrary, overexpression of the APN1 gene in Δyfh1 cells decreases spontaneous and induced mutagenesis. Our results show that frataxin deficiency in yeast cells leads to increased DNA base oxidation and requirement of Apn1 for repair, suggesting that DNA damage and repair could be important features in FA disease progression.

Molecular Mechanisms Underlying Genomic Instability in Brca-Deficient Cells

DTIC Science & Technology

2012-03-01

Fanconi anemia pathway for ICL repair. BRCA1 therefore has two separate roles in ICL repair that can be modulated by manipulating NHEJ, whereas FANCD2...repair pathway comprising at least 15 gene products. Mutation of any of these genes causes the human disease Fanconi anemia (FA), which is associated...genetic deficiency in components of the Fanconi anemia (FA) pathway (Wang, 2007). Cells from FA patients, or knockout mice with deficiencies in the FA

Formaldehyde exposure impairs the function and differentiation of NK cells.

PubMed

Kim, Eun-Mi; Lee, Hwa-Youn; Lee, Eun-Hee; Lee, Ki-Mo; Park, Min; Ji, Kon-Young; Jang, Ji-Hun; Jeong, Yun-Hwa; Lee, Kwang-Ho; Yoon, Il-Joo; Kim, Su-Man; Jeong, Moon-Jin; Kim, Kwang Dong; Kang, Hyung-Sik

2013-11-25

We investigated the cytotoxic effects of formaldehyde (FA) on lymphocytes. FA-exposed mice showed a profound reduction not only in the number of natural killer (NK) cells but also in the expression of NK cell-specific receptors, but these mice did not exhibit decreases in the numbers of T or B lymphocytes. FA exposure also induced decreases in NK cytolytic activity and in the expression of NK cell-associated genes, such as IFN-γ, perforin and CD122. To determine the effect of FA on tumorigenicity, C57BL/6 mice were subcutaneously injected with B16F10 melanoma cells after FA exposure. The mass of the B16F10 tumor and the concentration of extravascular polymorphonuclear leukocytes were greater than those in unexposed tumor-bearing control mice. The number and cytolytic activity of NK cells were also reduced in B16F10 tumor-bearing mice exposed to FA. To determine how FA reduces the NK cell number, NK precursor (pNK) cells were treated with FA, and the differentiation status of the NK cells was analyzed. NK cell differentiation was impaired by FA treatment in a concentration-dependent manner. These findings indicate that FA exposure may promote tumor progression by impairing NK cell function and differentiation. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Fluocinolone acetonide partially restores the mineralization of LPS-stimulated dental pulp cells through inhibition of NF-κB pathway and activation of AP-1 pathway

PubMed Central

Liu, Zhongning; Jiang, Ting; Wang, Xinzhi; Wang, Yixiang

2013-01-01

BACKGROUND AND PURPOSE Fluocinolone acetonide (FA) is commonly used as a steroidal anti-inflammatory drug. We recently found that in dental pulp cells (DPCs) FA has osteo-/odonto-inductive as well as anti-inflammatory effects. However, the mechanism by which FA induces these effects in DPCs is poorly understood. EXPERIMENTAL APPROACH The effect of FA on the mineralization of DPCs during inflammatory conditions and the underlying mechanism were investigated by real-time PCR, Western blot, EMSA, histochemical staining, immunostaining and pathway blockade assays. KEY RESULTS FA significantly inhibited the inflammatory response in LPS-treated DPCs not only by down-regulating the expression of pro–inflammation-related genes, but also by up-regulating the expression of the anti-inflammatory gene PPAR-γ and mineralization-related genes. Moreover, histochemical staining and immunostaining showed that FA could partially restore the expressions of alkaline phosphatase, osteocalcin and dentin sialophosphoprotein (DSPP) and mineralization in LPS-stimulated DPCs. Real-time PCR and Western blot analysis revealed that FA up-regulated DSPP and runt-related transcription factor 2 expression by inhibiting the expression of phosphorylated-NF-κB P65 and activating activator protein-1 (AP-1) (p-c-Jun and Fra-1). These results were further confirmed through EMSA, by detection of NF-κB DNA-binding activity and pathway blockade assays using a NF-κB pathway inhibitor, AP-1 pathway inhibitor and glucocorticoid receptor antagonist. CONCLUSIONS AND IMPLICATIONS Inflammation induced by LPS suppresses the mineralization process in DPCs. FA partially restored this osteo-/odonto-genesis process in LPS-treated DPCs and had an anti-inflammatory effect through inhibition of the NF-κB pathway and activation of the AP-1 pathway. Hence, FA is a potential new treatment for inflammation-associated bone/teeth diseases. PMID:24024985

Validation of General Electric Aviation DAPS2015 Digital Recording and 1/3 Octave Analysis System for Use in Support of Aircraft Noise Certification Efforts in Compliance with 14 CFR Part 36; Letter Report: V324-FA5JBQ-LR4 Rev. 1

DOT National Transportation Integrated Search

2017-10-31

NOTE: This is a revision of Letter Report V324-FA5JBQ-LR4. The purpose of the revision is to correctly identify the system designation for which the validation applies. The applicant originally identified the materials provided in support of this val...

Detecting blind building façades from highly overlapping wide angle aerial imagery

NASA Astrophysics Data System (ADS)

Burochin, Jean-Pascal; Vallet, Bruno; Brédif, Mathieu; Mallet, Clément; Brosset, Thomas; Paparoditis, Nicolas

2014-10-01

This paper deals with the identification of blind building façades, i.e. façades which have no openings, in wide angle aerial images with a decimeter pixel size, acquired by nadir looking cameras. This blindness characterization is in general crucial for real estate estimation and has, at least in France, a particular importance on the evaluation of legal permission of constructing on a parcel due to local urban planning schemes. We assume that we have at our disposal an aerial survey with a relatively high stereo overlap along-track and across-track and a 3D city model of LoD 1, that can have been generated with the input images. The 3D model is textured with the aerial imagery by taking into account the 3D occlusions and by selecting for each façade the best available resolution texture seeing the whole façade. We then parse all 3D façades textures by looking for evidence of openings (windows or doors). This evidence is characterized by a comprehensive set of basic radiometric and geometrical features. The blindness prognostic is then elaborated through an (SVM) supervised classification. Despite the relatively low resolution of the images, we reach a classification accuracy of around 85% on decimeter resolution imagery with 60 × 40 % stereo overlap. On the one hand, we show that the results are very sensitive to the texturing resampling process and to vegetation presence on façade textures. On the other hand, the most relevant features for our classification framework are related to texture uniformity and horizontal aspect and to the maximal contrast of the opening detections. We conclude that standard aerial imagery used to build 3D city models can also be exploited to some extent and at no additional cost for facade blindness characterisation.

Ferulic acid relaxed rat aortic, small mesenteric and coronary arteries by blocking voltage-gated calcium channel and calcium desensitization via dephosphorylation of ERK1/2 and MYPT1.

PubMed

Zhou, Zhong-Yan; Xu, Jia-Qi; Zhao, Wai-Rong; Chen, Xin-Lin; Jin, Yu; Tang, Nuo; Tang, Jing-Yi

2017-11-15

Ferulic acid, a natural ingredient presents in several Chinese Materia Medica such as Radix Angelicae Sinensis, has been identified as an important multifunctional and physiologically active small molecule. However, its pharmacological activity in different blood vessel types and underlying mechanisms are unclear. The present study was to investigate the vascular reactivity and the possible action mechanism of FA on aorta, small mesenteric arteries and coronary arteries isolated from Wistar rats. We found FA dose-dependently relieved the contraction of aorta, small mesenteric arteries and coronary arteries induced by different contractors, U46619, phenylephrine (Phe) and KCl. The relaxant effect of FA was not affected by L-NAME (eNOS inhibitor), ODQ (soluble guanylate cyclase inhibitor), and mechanical removal of endothelium in thoracic aortas. The contraction caused by 60mM KCl (60K) was concentration-dependently hindered by FA pretreatment in all three types of arteries. In Ca 2+ -free 60K solution, FA weakened Ca 2+ -related contraction in a concentration dependent manner. And FA relaxed both fluoride and phorbol ester which were PKC, ERK and Rho-kinase activators induced contraction in aortic rings with or without Ca 2+ in krebs solution. Western blotting experiments in A7r5 cells revealed that FA inhibited calcium sensitization via dephosphorylation of ERK1/2 and MYPT1. Furthermore, the relaxation effect of FA was attenuated by verapamil (calcium channel blocker), ERK inhibitor, and fasudil (ROCK inhibitor). These results provide evidence that FA exhibits endothelium-independent vascular relaxant effect in different types of arteries. The molecular mechanism of vasorelaxation activity of FA probably involved calcium channel inhibition and calcium desensitization. Copyright © 2017. Published by Elsevier B.V.

microRNA-188 is downregulated in oral squamous cell carcinoma and inhibits proliferation and invasion by targeting SIX1.

PubMed

Wang, Lili; Liu, Hongchen

2016-03-01

microRNA-188 expression is downregulated in several tumors. However, its function and mechanism in human oral squamous cell carcinoma (OSCC) remains obscure. The present study aims to identify the expression pattern, biological roles, and potential mechanism by which miR-188 dysregulation is associated with oral squamous cell carcinoma. Significant downregulation of miR-188 was observed in OSCC tissues compared with paired normal tissues. In vitro, gain-of-function, loss-of-function experiments were performed to examine the impact of miR-188 on cancer cell proliferation, invasion, and cell cycle progression. Transfection of miR-188 mimics suppressed Detroit 562 cell proliferation, cell cycle progression and invasion, with downregulation of cyclin D1, MMP9, and p-ERK. Transfection of miR-188 inhibitor in FaDu cell line with high endogenous expression exhibited the opposite effects. Using fluorescence reporter assays, we confirmed that SIX1 was a direct target of miR-188 in OSCC cells. Transfection of miR-188 mimics downregulated SIX1 expression. SIX1 siRNA treatment abrogated miR-188 inhibitor-induced cyclin D1 and MMP9 upregulation. In addition, we found that SIX1 was overexpressed in 32 of 80 OSCC tissues. In conclusion, this study indicates that miR-188 downregulation might be associated with oral squamous cell carcinoma progression. miR-188 suppresses proliferation and invasion by targeting SIX1 in oral squamous cell carcinoma cells.

Dietary fructans, but not cellulose, decrease triglyceride accumulation in the liver of obese Zucker fa/fa rats.

PubMed

Daubioul, Catherine; Rousseau, Nicolas; Demeure, Roger; Gallez, Bernard; Taper, Henryk; Declerck, Barbara; Delzenne, Nathalie

2002-05-01

This study was designed to compare the effects of dietary supplementation with nondigestible carbohydrates, differing in fermentability by colonic bacteria, on hepatic steatosis in growing obese Zucker rats. Male Zucker fa/fa rats were divided into three groups: a control group that received the basal diet, a fructan group that received 10 g highly fermented Synergy 1/100 g diet and a cellulose group that received 10 g poorly fermented Vivapur Microcrystalline cellulose/100 g diet. Rats consuming fructan had a lower energy intake, a lower body weight and less triacylglycerol accumulation in the liver as assessed in vivo by nuclear magnetic resonance (NMR) spectroscopy, and ex vivo by biochemical and histochemical analysis compared with the control and/or cellulose groups. The high fermentation of fructans compared with cellulose was reflected by greater cecal contents and by a twofold greater propionate concentration in the portal vein of rats fed fructan compared with those fed cellulose. By measuring the capacity of hepatocytes isolated from liver of Zucker rats to synthesize triglycerides or total lipids from different precursors, we showed that propionate, at the concentrations measured in the portal vein of rats treated with fructan, selectively decreased the incorporation of acetate into total lipids, a phenomenon that could contribute, along with the lower energy intake, to less triglyceride accumulation in the liver of obese Zucker rats fed dietary fructans.

Optimizing Maintenance Manpower for USMC F/A-18 Squadrons

DTIC Science & Technology

2016-06-01

experience level, with the requirement of keeping a standard number of aircraft operationally ready. MVP results show areas of deficit, either manpower ...MAINTENANCE MANPOWER FOR USMC F/A-18 SQUADRONS by Kevin J. Goodwin June 2016 Thesis Co-Advisors: W. Matthew Carlyle Robert F. Dell Second...REPORT TYPE AND DATES COVERED Master’s thesis 4. TITLE AND SUBTITLE OPTIMIZING MAINTENANCE MANPOWER FOR USMC F/A-18 SQUADRONS 5. FUNDING NUMBERS 6

Toxicity of cosmetic preservatives on human ocular surface and adnexal cells.

PubMed

Chen, Xiaomin; Sullivan, David A; Sullivan, Amy Gallant; Kam, Wendy R; Liu, Yang

2018-05-01

Cosmetic products, such as mascara, eye shadow, eyeliner and eye makeup remover are used extensively to highlight the eyes or clean the eyelids, and typically contain preservatives to prevent microbial growth. These preservatives include benzalkonium chloride (BAK) and formaldehyde (FA)-releasing preservatives. We hypothesize that these preservatives, at concentrations (BAK = 1 mg/ml; FA = 0.74 mg/ml) approved for consumer use, are toxic to human ocular surface and adnexal cells. Accordingly, we tested the influence of BAK and FA on the morphology, survival, and proliferation and signaling ability of immortalized human meibomian gland (iHMGECs), corneal (iHCECs) and conjunctival (iHConjECs) epithelial cells. iHMGECs, iHCECs and iHConjECs were cultured with different concentrations of BAK (5 μg/ml to 0.005 μg/ml) or FA (1 mg/ml to 1 μg/ml) under basal, proliferating or differentiating conditions up to 7 days. We used low BAK levels, because we found that 0.5 mg/ml and 50 μg/ml BAK killed iHMGECs within 1 day after a 15 min exposure. Experimental procedures included analyses of cell appearance, cell number, and neutral lipid content (LipidTox), lysosome accumulation (LysoTracker) and AKT signaling in all 3 cell types. Our results demonstrate that BAK and FA cause dose-dependent changes in the morphology, survival, proliferation and AKT signaling of iHMGECs, iHCECs and iHConjECs. Many of the concentrations tested induced cell atrophy, poor adherence, decreased proliferation and death, after 5 days of exposure. Cellular signaling, as indicated by AKT phosphorylation after 15 (FA) or 30 (BAK) minutes of treatment, was also reduced in a dose-dependent fashion in all 3 cell types, irrespective of whether cells had been cultured under proliferating or differentiating conditions. Our results support our hypothesis and demonstrate that the cosmetic preservatives, BAK and FA, exert many toxic effects on cells of the ocular surface and adnexa

The Fanconi Anemia C Protein Binds to and Regulates Stathmin-1 Phosphorylation

PubMed Central

Magron, Audrey; Elowe, Sabine; Carreau, Madeleine

2015-01-01

The Fanconi anemia (FA) proteins are involved in a signaling network that assures the safeguard of chromosomes. To understand the function of FA proteins in cellular division events, we investigated the interaction between Stathmin-1 (STMN1) and the FA group C (FANCC) protein. STMN1 is a ubiquitous cytosolic protein that regulates microtubule dynamics. STMN1 activities are regulated through phosphorylation-dephosphorylation mechanisms that control assembly of the mitotic spindle, and dysregulation of STMN1 phosphorylation is associated with mitotic aberrancies leading to chromosome instability and cancer progression. Using different biochemical approaches, we showed that FANCC interacts and co-localizes with STMN1 at centrosomes during mitosis. We also showed that FANCC is required for STMN1 phosphorylation, as mutations in FANCC reduced serine 16- and 38-phosphorylated forms of STMN1. Phosphorylation of STMN1 at serine 16 is likely an event dependent on a functional FA pathway, as it is reduced in FANCA- and FANCD2-mutant cells. Furthermore, FA-mutant cells exhibited mitotic spindle anomalies such as supernumerary centrosomes and shorter mitotic spindles. These results suggest that FA proteins participate in the regulation of cellular division via the microtubule-associated protein STMN1. PMID:26466335

Se@SiO2-FA-CuS nanocomposites for targeted delivery of DOX and nano selenium in synergistic combination of chemo-photothermal therapy.

PubMed

Wang, Yeying; Liu, Xijian; Deng, Guoying; Sun, Jian; Yuan, Haikuan; Li, Qi; Wang, Qiugeng; Lu, Jie

2018-02-08

In this study, a versatile tumor-targeted and multi-stimuli-responsive drug delivery vehicle (Se particle@porous silica-folic acid-copper sulfide/doxorubicin (Se@SiO 2 -FA-CuS/DOX)) was fabricated for combined photothermal therapy with chemotherapy in cancer treatment. Due to excellent targeting ability, the Se@SiO 2 -FA-CuS/DOX nanocomposites actively accumulated in tumor tissues and thus provided photothermal therapy under NIR irradiation and chemotherapy through the release of DOX and Se. Owing to the synergistic effect of chemotherapy (Se and DOX) and photothermal therapy, the Se@SiO 2 -FA-CuS/DOX nanocomposites could efficiently inhibit cancer cells both in vitro and in vivo and even completely eliminate tumors. Moreover, as the toxicity of DOX could be reduced by Se, the treatment using Se@SiO 2 -FA-CuS/DOX nanocomposites exhibited no appreciable adverse reactions. Thus, the Se@SiO 2 -FA-CuS/DOX nanocomposites have great potential as a multifunctional nanoplatform in future clinical applications.

Identifying microRNAs that Regulate Neuroblastoma Cell Differentiation

DTIC Science & Technology

2015-10-01

Award Number: W81XWH-13-1-0241 TITLE: Identifying that Regulate Neuroblastoma Cell Differentiation PRINCIPAL INVESTIGATOR: Dr. Liqin Du...inducing miRNA, miR- 449a. We examined the differentiation-inducing function of miR-449a in multiple neuroblastoma cell lines. We have demonstrated that...miR-449a functions as an inducer of cell differentiation in neuroblastoma cell lines with distinct genetic backgrounds, including the MYCN

ciliaFA: a research tool for automated, high-throughput measurement of ciliary beat frequency using freely available software

PubMed Central

2012-01-01

Background Analysis of ciliary function for assessment of patients suspected of primary ciliary dyskinesia (PCD) and for research studies of respiratory and ependymal cilia requires assessment of both ciliary beat pattern and beat frequency. While direct measurement of beat frequency from high-speed video recordings is the most accurate and reproducible technique it is extremely time consuming. The aim of this study was to develop a freely available automated method of ciliary beat frequency analysis from digital video (AVI) files that runs on open-source software (ImageJ) coupled to Microsoft Excel, and to validate this by comparison to the direct measuring high-speed video recordings of respiratory and ependymal cilia. These models allowed comparison to cilia beating between 3 and 52 Hz. Methods Digital video files of motile ciliated ependymal (frequency range 34 to 52 Hz) and respiratory epithelial cells (frequency 3 to 18 Hz) were captured using a high-speed digital video recorder. To cover the range above between 18 and 37 Hz the frequency of ependymal cilia were slowed by the addition of the pneumococcal toxin pneumolysin. Measurements made directly by timing a given number of individual ciliary beat cycles were compared with those obtained using the automated ciliaFA system. Results The overall mean difference (± SD) between the ciliaFA and direct measurement high-speed digital imaging methods was −0.05 ± 1.25 Hz, the correlation coefficient was shown to be 0.991 and the Bland-Altman limits of agreement were from −1.99 to 1.49 Hz for respiratory and from −2.55 to 3.25 Hz for ependymal cilia. Conclusions A plugin for ImageJ was developed that extracts pixel intensities and performs fast Fourier transformation (FFT) using Microsoft Excel. The ciliaFA software allowed automated, high throughput measurement of respiratory and ependymal ciliary beat frequency (range 3 to 52 Hz) and avoids operator error due to selection bias. We have

The nuclease hSNM1B/Apollo is linked to the Fanconi anemia pathway via its interaction with FANCP/SLX4.

PubMed

Salewsky, Bastian; Schmiester, Maren; Schindler, Detlev; Digweed, Martin; Demuth, Ilja

2012-11-15

The recessive genetic disorder Fanconi anemia (FA) is clinically characterized by congenital defects, bone marrow failure and an increased incidence of cancer. Cells derived from FA patients exhibit hypersensitivity to DNA interstrand crosslink (ICL)-inducing agents. We have earlier reported a similar cellular phenotype for human cells depleted of hSNM1B/Apollo (siRNA). In fact, hSNM1B/Apollo has a dual role in the DNA damage response and in generation and maintenance of telomeres, the latter function involving interaction with the shelterin protein TRF2. Here we find that ectopically expressed hSNM1B/Apollo co-immunoprecipitates with SLX4, a protein recently identified as a new FA protein, FANCP, and known to interact with several structure-specific nucleases. As shown by immunofluorescence analysis, FANCP/SLX4 depletion (siRNA) resulted in a significant reduction of hSNM1B/Apollo nuclear foci, supporting the functional relevance of this new protein interaction. Interestingly, as an additional consequence of FANCP/SLX4 depletion, we found a reduction of cellular TRF2, in line with its telomere-related function. Finally, analysis of human cells following double knockdown of hSNM1B/Apollo and FANCP/SLX4 indicated that they function epistatically. These findings further substantiate the role of hSNM1B/Apollo in a downstream step of the FA pathway during the repair of DNA ICLs.

IL-6, IL-8, MMP-2, MMP-9 are overexpressed in Fanconi anemia cells through a NF-κB/TNF-α dependent mechanism.

PubMed

Epanchintsev, Alexey; Shyamsunder, Pavithra; Verma, Rama S; Lyakhovich, Alex

2015-12-01

Fanconi anemia (FA) is a rare autosomal recessive genetic disorder associated with a bone-marrow failure, genome instability, hypersensitivity to DNA crosslinking agents and a predisposition to cancer. Mutations have been documented in 16 FA genes that participate in the FA-BRCA DNA repair pathway, a fundamental pathway in the development of the disease and the presentation of its symptoms. FA cells have been characterized by an overproduction of cytokines, MAPKs, and Interleukins. Through this study we have identified the overexpression of additional secretory factors such as IL-6, IL-8, MMP-2, and MMP-9 in FA cells and in cells depleted of FANCA or FANCC and proved that their expression is under the control of NF-κB/TNF-α signaling pathways. We also demonstrated that these overexpressed secretory factors were effective in promoting the proliferation, migration, and invasion of surrounding tumor cells a fundamental event in the process of epithelial mesenchymal transition (EMT) and that they also modulated the expression of EMT markers such as E-cadherin and SNAIL. Overall our data suggest that the upregulation of EMT promoting factors in FA may contribute to predisposing FA patients to cancer, thereby providing new insights into possible therapeutic interventions. © 2014 Wiley Periodicals, Inc.

Free-to-Roll Investigation of the Pre-Production F/A-18E Powered Approach Wing Drop

NASA Technical Reports Server (NTRS)

Owens, D. Bruce; Bryant, Elaine M.; Barlow, Jewel B.

2005-01-01

A free-to-roll study of the low-speed lateral characteristics of the pre-production F/A-18E was conducted in the NASA Langley 12-Foot Low-Speed Tunnel. In developmental flight tests the F/A-18E unexpectedly experienced uncommanded lateral motions in the power approach configuration. The objective of this study was to determine the feasibility of using the free-to-roll technique for the detection of uncommanded lateral motions for the pre-production F/A-18E in the power approach configuration. The data revealed that this technique in conjunction with static data revealed insight into the cause of the lateral motions. The free-to-roll technique identified uncommanded lateral motions at the same angle-of-attack range as experienced in flight tests. The cause of the uncommanded lateral motions was unsteady asymmetric wing stall. The paper also shows that free-to-roll data or static force and moment data alone are not enough to accurately capture the potential for an aircraft to experience uncommanded lateral motion.

Proteomic profiling identifies the inorganic pyrophosphatase (PPA1) protein as a potential biomarker of metastasis in laryngeal squamous cell carcinoma.

PubMed

Bodnar, Magdalena; Luczak, Magdalena; Bednarek, Kinga; Szylberg, Lukasz; Marszalek, Andrzej; Grenman, Reidar; Szyfter, Krzysztof; Jarmuz-Szymczak, Malgorzata; Giefing, Maciej

2016-06-01

Relapse and metastasis are the main causes of unfavorable outcome in head and neck cancers. Whereas, understanding of the molecular background of these processes is far from being complete. Therefore, in this study we aimed to identify potential biomarker candidates of relapse and metastasis in laryngeal squamous cell carcinoma (LSCC) by combining the 2D electrophoresis based protein screen and immunohistochemical analysis of candidate proteins. We screened three groups of LSCC cell lines derived from primary tumors, recurrent tumors and metastases and identified seven proteins that differed significantly in relative abundance between the analyzed groups. Among the identified proteins were the heat shock proteins HSP60 and HSP70 that were significantly downregulated both in recurrences- and metastases-derived cell lines but not in primary tumor-derived cell lines. Moreover, we identified significant upregulation of the annexin V, calreticulin and the inorganic pyrophosphatase (PPA1) exclusively in the metastases-derived cell lines. As these upregulated proteins could potentially become novel biomarkers of metastasis, we have compared their abundance in primary tumor LSCC N(0) cases, primary tumor LSCC N(+) cases as well as in LSCC metastases N(+). Our results show an intense increase of cytoplasmic PPA1 abundance in the N(+) (p = 0.000042) compared to the N(0) group. In summary, we show a group of proteins deregulated in recurrences and metastases of LSCC. Moreover, we suggest the PPA1 protein as a potential new biomarker for metastasis in this cancer.

The fa2 gene and molecular markers mapping in the gp segment of the Pisum linkage group V.

PubMed

Gawłowska, M; Święcicki, W

2016-08-01

Review studies on the world Pisum genetic resources have shown that stem fasciation is controlled by three loci, i.e., fa1 (LGIV; Wt 10006 - type line of the Polish Gene Bank), fa2 (LGV, the line Wt 12185), and fas (LGIII, the line Shtambovii). Outstanding advantages of this character (e.g., pods gathered in upper part of a stem) resulted in breeding some cultivars. Preliminary investigations suggested linkages of the newly described fa2 gene within the gp-U segment. Based on the further linkage test crosses, it was stated that the fa2 is localized between the gp and Pis_Gen_9_3_1 markers (in the LGV). Additionally, four molecular markers (AD175, AB146, AC58, and AD280) and the morphological marker lk were also localized in this segment. Moreover, rms5, lum3, and cri were found to map on the other side of gp with tight linkage observed between lum3 and cri.

A high-throughput screen for inhibitors of the prolyl isomerase, Pin1, identifies a seaweed polyphenol that reduces adipose cell differentiation.

PubMed

Mori, Tadashi; Hidaka, Masafumi; Ikuji, Hiroko; Yoshizawa, Ibuki; Toyohara, Haruhiko; Okuda, Toru; Uchida, Chiyoko; Asano, Tomoichiro; Yotsu-Yamashita, Mari; Uchida, Takafumi

2014-01-01

The peptidyl prolyl cis/trans isomerase Pin1 enhances the uptake of triglycerides and the differentiation of fibroblasts into adipose cells in response to insulin stimulation. Pin1 downregulation could be a potential approach to prevent and treat obesity-related disorders. In order to identify an inhibitor of Pin1 that exhibited minimal cytotoxicity, we established a high-throughput screen for Pin1 inhibitors and used this method to identify an inhibitor from 1,056 crude fractions of two natural product libraries. The candidate, a phlorotannin called 974-B, was isolated from the seaweed, Ecklonia kurome. 974-B inhibited the differentiation of mouse embryonic fibroblasts and 3T3-L1 cells into adipose cells without inducing cytotoxicity. We discovered the Pin1 inhibitor, 974-B, from the seaweed, E. kurome, and showed that it blocks the differentiation of fibroblasts into adipose cells, suggesting that 974-B could be a lead drug candidate for obesity-related disorders.

Plekhs1 and Prdx3 are candidate genes responsible for mild hyperglycemia associated with obesity in a new animal model of F344-fa-nidd6 rat.

PubMed

Kotoh, Jun; Sasaki, Daiki; Matsumoto, Kozo; Maeda, Akihiko

2016-12-01

Type 2 diabetes is a polygenic disease and characterized by hyperglycemia and insulin resistance, and it is strongly associated with obesity. However, the mechanism by which obesity contributes to onset of type 2 diabetes is not well understood. We generated rat strains with a hyperglycemic quantitative trait locus (QTL) derived from the Otsuka Long-Evans Tokushima Fatty rat and a fa/fa (Lepr -/- ) locus derived from the Zucker Fatty rat. Phenotypes for plasma glucose, and insulin levels were measured, and RNA and protein levels were determined using reverse transcription quantitative PCR and Western blot analyses, respectively. Compared with the obese control strain F344-fa (Lepr -/- ), plasma glucose levels of the obese F344-fa-nidd6 (Lepr -/- and Nidd6/of) significantly increased, and plasma insulin levels significantly decreased. These phenotypes were not observed in the lean strains, suggesting that the Nidd6/of locus harbors a diabetogenic gene associated with obesity. We measured the expression of 41 genes in the Nidd6/of QTL region of each strain and found that the mRNA expression levels of the two genes significantly differed between the obese strains. The two genes, pleckstrin homology domain-containing, family S member 1 (Plechs1) and peroxiredoxin III (Prdx3), were differentially expressed only in the obese rats, suggesting that these two genes are involved in the mild elevation of blood glucose levels and insulin resistance in obesity.

Biotransformation of 2,4,6,8,10,12-Hexanitro-2,4,6,8,10,12-Hexaazaisowurtzitane (CL-20) by Denitrifying Pseudomonas sp. Strain FA1

PubMed Central

Bhushan, Bharat; Paquet, Louise; Spain, Jim C.; Hawari, Jalal

2003-01-01

The microbial and enzymatic degradation of a new energetic compound, 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20), is not well understood. Fundamental knowledge about the mechanism of microbial degradation of CL-20 is essential to allow the prediction of its fate in the environment. In the present study, a CL-20-degrading denitrifying strain capable of utilizing CL-20 as the sole nitrogen source, Pseudomonas sp. strain FA1, was isolated from a garden soil. Studies with intact cells showed that aerobic conditions were required for bacterial growth and that anaerobic conditions enhanced CL-20 biotransformation. An enzyme(s) involved in the initial biotransformation of CL-20 was shown to be membrane associated and NADH dependent, and its expression was up-regulated about 2.2-fold in CL-20-induced cells. The rates of CL-20 biotransformation by the resting cells and the membrane-enzyme preparation were 3.2 ± 0.1 nmol h−1 mg of cell biomass−1 and 11.5 ± 0.4 nmol h−1 mg of protein−1, respectively, under anaerobic conditions. In the membrane-enzyme-catalyzed reactions, 2.3 nitrite ions (NO2−), 1.5 molecules of nitrous oxide (N2O), and 1.7 molecules of formic acid (HCOOH) were produced per reacted CL-20 molecule. The membrane-enzyme preparation reduced nitrite to nitrous oxide under anaerobic conditions. A comparative study of native enzymes, deflavoenzymes, and a reconstituted enzyme(s) and their subsequent inhibition by diphenyliodonium revealed that biotransformation of CL-20 is catalyzed by a membrane-associated flavoenzyme. The latter catalyzed an oxygen-sensitive one-electron transfer reaction that caused initial N denitration of CL-20. PMID:12957905

Genetic Analysis of Strawberry Fruit Aroma and Identification of O-Methyltransferase FaOMT as the Locus Controlling Natural Variation in Mesifurane Content1[C][W][OA

PubMed Central

Zorrilla-Fontanesi, Yasmín; Rambla, José-Luis; Cabeza, Amalia; Medina, Juan J.; Sánchez-Sevilla, José F.; Valpuesta, Victoriano; Botella, Miguel A.; Granell, Antonio; Amaya, Iraida

2012-01-01

Improvement of strawberry (Fragaria × ananassa) fruit flavor is an important goal in breeding programs. To investigate genetic factors controlling this complex trait, a strawberry mapping population derived from genotype ‘1392’, selected for its superior flavor, and ‘232’ was profiled for volatile compounds over 4 years by headspace solid phase microextraction coupled to gas chromatography and mass spectrometry. More than 300 volatile compounds were detected, of which 87 were identified by comparison of mass spectrum and retention time to those of pure standards. Parental line ‘1392’ displayed higher volatile levels than ‘232’, and these and many other compounds with similar levels in both parents segregated in the progeny. Cluster analysis grouped the volatiles into distinct chemically related families and revealed a complex metabolic network underlying volatile production in strawberry fruit. Quantitative trait loci (QTL) detection was carried out over 3 years based on a double pseudo-testcross strategy. Seventy QTLs covering 48 different volatiles were detected, with several of them being stable over time and mapped as major QTLs. Loci controlling γ-decalactone and mesifurane content were mapped as qualitative traits. Using a candidate gene approach we have assigned genes that are likely responsible for several of the QTLs. As a proof of concept we show that one homoeolog of the O-methyltransferase gene (FaOMT) is the locus responsible for the natural variation of mesifurane content. Sequence analysis identified 30 bp in the promoter of this FaOMT homoeolog containing putative binding sites for basic/helix-loop-helix, MYB, and BZIP transcription factors. This polymorphism fully cosegregates with both the presence of mesifurane and the high expression of FaOMT during ripening. PMID:22474217

A High-Throughput Genetic Complementation Assay in Yeast Cells Identified Selective Inhibitors of Sphingosine Kinase 1 Not Found Using a Cell-Free Enzyme Assay.

PubMed

Kashem, Mohammed A; Kennedy, Charles A; Fogarty, Kylie E; Dimock, Janice R; Zhang, Yunlong; Sanville-Ross, Mary L; Skow, Donna J; Brunette, Steven R; Swantek, Jennifer L; Hummel, Heidi S; Swindle, John; Nelson, Richard M

2016-01-01

Sphingosine kinase 1 (SphK1) is a lipid kinase that phosphorylates sphingosine to produce the bioactive sphingolipid, sphingosine-1-phosphate (S1P), and therefore represents a potential drug target for a variety of pathological processes such as fibrosis, inflammation, and cancer. We developed two assays compatible with high-throughput screening to identify small-molecule inhibitors of SphK1: a purified component enzyme assay and a genetic complementation assay in yeast cells. The biochemical enzyme assay measures the phosphorylation of sphingosine-fluorescein to S1P-fluorescein by recombinant human full-length SphK1 using an immobilized metal affinity for phosphochemicals (IMAP) time-resolved fluorescence resonance energy transfer format. The yeast assay employs an engineered strain of Saccharomyces cerevisiae, in which the human gene encoding SphK1 replaced the yeast ortholog and quantitates cell viability by measuring intracellular adenosine 5'-triphosphate (ATP) using a luciferase-based luminescent readout. In this assay, expression of human SphK1 was toxic, and the resulting yeast cell death was prevented by SphK1 inhibitors. We optimized both assays in a 384-well format and screened ∼10(6) compounds selected from the Boehringer Ingelheim library. The biochemical IMAP high-throughput screen identified 5,561 concentration-responsive hits, most of which were ATP competitive and not selective over sphingosine kinase 2 (SphK2). The yeast screen identified 205 concentration-responsive hits, including several distinct compound series that were selective against SphK2 and were not ATP competitive.

TRIM.FaTE Public Reference Library Documentation

EPA Pesticide Factsheets

TRIM.FaTE is a spatially explicit, compartmental mass balance model that describes the movement and transformation of pollutants over time, through a user-defined, bounded system that includes both biotic and abiotic compartments.

Role of WDHD1 in Human Papillomavirus-Mediated Oncogenesis Identified by Transcriptional Profiling of E7-Expressing Cells

PubMed Central

Zhou, Yunying; Zhang, Qishu; Gao, Ge; Zhang, Xiaoli; Liu, Yafei; Yuan, Shoudao

2016-01-01

-HPV agents are not available. In this study, we performed RNA-seq to characterize transcriptional profiling of keratinocytes expressing HPV-16 E7 and identified more than 200 genes that were differentially expressed between E7 and vector control cells. Through bioinformatics analysis, pathways altered in E7-expressing cells were identified. Significantly, the WDHD1 gene, one of the genes that is upregulated in E7-expressing cells, was found to play an important role in E7-induced G1 checkpoint abrogation and rereplication. These studies shed light on mechanisms by which HPV induces genomic instability and have therapeutic implications. PMID:27099318

Lung Cancer Cell Line Screen Links Fanconi Anemia/BRCA Pathway Defects to Increased Relative Biological Effectiveness of Proton Radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Qi; Ghosh, Priyanjali; Magpayo, Nicole

2015-04-01

Purpose: Growing knowledge of genomic heterogeneity in cancer, especially when it results in altered DNA damage responses, requires re-examination of the generic relative biological effectiveness (RBE) of 1.1 of protons. Methods and Materials: For determination of cellular radiosensitivity, we irradiated 17 lung cancer cell lines at the mid-spread-out Bragg peak of a clinical proton beam (linear energy transfer, 2.5 keV/μm). For comparison, 250-kVp X rays and {sup 137}Cs γ-rays were used. To estimate the RBE of protons relative to {sup 60}Co (Co60eq), we assigned an RBE(Co60Eq) of 1.1 to X rays to correct the physical dose measured. Standard DNA repair foci assaysmore » were used to monitor damage responses. FANCD2 was depleted using RNA interference. Results: Five lung cancer cell lines (29.4%) exhibited reduced clonogenic survival after proton irradiation compared with X-irradiation with the same physical doses. This was confirmed in a 3-dimensional sphere assay. Corresponding proton RBE(Co60Eq) estimates were statistically significantly different from 1.1 (P≤.05): 1.31 to 1.77 (for a survival fraction of 0.5). In 3 of these lines, increased RBE was correlated with alterations in the Fanconi anemia (FA)/BRCA pathway of DNA repair. In Calu-6 cells, the data pointed toward an FA pathway defect, leading to a previously unreported persistence of proton-induced RAD51 foci. The FA/BRCA-defective cells displayed a 25% increase in the size of subnuclear 53BP1 foci 18 hours after proton irradiation. Conclusions: Our cell line screen has revealed variations in proton RBE that are partly due to FA/BRCA pathway defects, suggesting that the use of a generic RBE for cancers should be revisited. We propose that functional biomarkers, such as size of residual 53BP1 foci, may be used to identify cancers with increased sensitivity to proton radiation.« less

Generation of reactive oxygen species by grape seed extract causes irreparable DNA damage leading to G2/M arrest and apoptosis selectively in head and neck squamous cell carcinoma cells.

PubMed

Shrotriya, Sangeeta; Deep, Gagan; Gu, Mallikarjuna; Kaur, Manjinder; Jain, Anil K; Inturi, Swetha; Agarwal, Rajesh; Agarwal, Chapla

2012-04-01

Head and neck squamous cell carcinoma (HNSCC) accounts for 6% of all malignancies in USA and unfortunately the recurrence of secondary primary tumors and resistance against conventional treatments decrease the overall 5 year survival rate in HNSCC patients. Thus, additional approaches are needed to control HNSCC. Here, for the first time, employing human HNSCC Detroit 562 and FaDu cells as well as normal human epidermal keratinocytes, we investigate grape seed extract (GSE) efficacy and associated mechanism in both cell culture and nude mice xenografts. GSE selectively inhibited the growth and caused cell cycle arrest and apoptotic death in both Detroit 562 and FaDu cells by activating DNA damage checkpoint cascade, including ataxia telangiectasia mutated/ataxia telangiectasia-Rad3-related-checkpoint kinase 1/2-cell division cycle 25C as well as caspases 8, 9 and 3. Consistent with these results, GSE treatment resulted in a strong DNA damage and a decrease in the levels of DNA repair molecules breast cancer gene 1 and Rad51 and DNA repair foci. GSE-caused accumulation of intracellular reactive oxygen species was identified as a major mechanism of its effect for growth inhibition, DNA damage and apoptosis, which was remarkably reversed by antioxidant N-acetylcysteine. GSE feeding to nude mice decreased Detroit 562 and FaDu xenograft tumor growth by 67 and 65% (P < 0.001), respectively. In immunohistochemical analysis, xenografts from GSE-fed groups showed decreased proliferation but increased DNA damage and apoptosis. Together, these findings show that GSE targets both DNA damage and repair and provide mechanistic insights for its efficacy selectively against HNSCC both in cell culture and mouse xenograft, supporting its translational potential against HNSCC.

Color Vision and the Railways: Part 3. Comparison of FaLant, OPTEC 900, and Railway LED Lantern Tests.

PubMed

Dain, Stephen J; Casolin, Armand; Long, Jennifer

2015-02-01

The Farnsworth Lantern (FaLant) and the OPTEC 900 are nominated in the Commission Internationale de l'Éclairage (CIE) Color Vision Standard 2. Neither test uses the railway signal color code of red, yellow, and green, and only the OPTEC 900 is commercially available. The Railway LED Lantern Test (RLLT) is based on railway signaling practices in New South Wales, Australia, and is nominated in the Australian railway medical standard. The objective of this study is to compare the performance of the three lantern tests. The RLLT, FaLant, and OPTEC 900 were administered to 46 color vision-normal and 37 color vision-deficient (CVD) subjects. The pattern of errors on the RLLT was different from that of the FaLant and OPTEC 900. This may be accounted for, at least in part, by the different colors and the use of blank presentations in the RLLT. The three lanterns showed agreement in failing 21 and passing 6 of the CVD subjects (72.9%). The lanterns gave different results for 10 CVD subjects (27.9%): n = 5 passed only the RLLT and n = 3 passed only the FaLant; n = 1 failed only the FaLant and n = 1 failed only the RLLT. The overall failure rate by CVD for each lantern was 67.6% (RLLT), 73.0% (FaLant), and 78.4% (OPTEC 900). Despite the different construction principles, the pass/fail levels of the RLLT, FaLant, and OPTEC 900 are comparable and consistent with the performance of other lanterns listed by the CIE for Color Vision Standard 2. The RLLT may be a little easier to pass and is based on the signal color code used and actual signaling practice. We propose that the RLLT is also an appropriate lantern for CIE Color Vision Standard 2.

Doxorubicin-conjugated D-glucosamine- and folate- bi-functionalised InP/ZnS quantum dots for cancer cells imaging and therapy.

PubMed

Ranjbar-Navazi, Zahra; Eskandani, Morteza; Johari-Ahar, Mohammad; Nemati, Ali; Akbari, Hamid; Davaran, Soudabeh; Omidi, Yadollah

2018-03-01

Nanoscaled quantum dots (QDs), with unique optical properties have been used for the development of theranostics. Here, InP/ZnS QDs were synthesised and functionalised with folate (QD-FA), D-glucosamine (QD-GA) or both (QD-FA-GA). The bi-functionalised QDs were further conjugated with doxorubicin (QD-FA-GA-DOX). Optimum Indium to fatty acid (In:MA) ratio was 1:3.5. Transmission electron microscopy (TEM) micrographs revealed spherical morphology for the QDs (11 nm). Energy-dispersive spectroscopy (EDS) spectrum confirmed the chemical composition of the QDs. MTT analysis in the OVCAR-3 cells treated with bare QDs, QD-FA, QD-GA, QD-FA-GA and QD-FA-GA-DOX (0.2 mg/mL of QDs) after 24 h indicated low toxicity for the bare QDs and functionalised QDs (about 80-90% cell viability). QD-FA-GA-DOX nanoparticles elicited toxicity in the cells. Cellular uptake of the engineered QDs were investigated in both folate receptor (FR)-positive OVCAR-3 cells and FR-negative A549 cells using fluorescence microscopy and FACS flow cytometry. The FA-functionalised QDs showed significantly higher uptake in the FR-positive OVCAR-3 cells, nonetheless the GA-functionalised QDs resulted in an indiscriminate uptake in both cell lines. In conclusion, our findings indicated that DOX-conjugated FA-armed QDs can be used as theranostics for simultaneous imaging and therapy of cancer.

pH-sensitive and folic acid-targeted MPEG-PHIS/FA-PEG-VE mixed micelles for the delivery of PTX-VE and their antitumor activity.

PubMed

Di, Yan; Li, Ting; Zhu, Zhihong; Chen, Fen; Jia, Lianqun; Liu, Wenbing; Gai, Xiumei; Wang, Yingying; Pan, Weisan; Yang, Xinggang

2017-01-01

The aim of this study was to simultaneously introduce pH sensitivity and folic acid (FA) targeting into a micelle system to achieve quick drug release and to enhance its accumulation in tumor cells. Paclitaxel-(+)-α-tocopherol (PTX-VE)-loaded mixed micelles (PHIS/FA/PM) fabricated by poly(ethylene glycol) methyl ether-poly(histidine) (MPEG-PHIS) and folic acid-poly(ethylene glycol)-(+)-α-tocopherol (FA-PEG-VE) were characterized by dynamic light scattering and transmission electron microscopy (TEM). The mixed micelles had a spherical morphology with an average diameter of 137.0±6.70 nm and a zeta potential of -48.7±4.25 mV. The drug encapsulation and loading efficiencies were 91.06%±2.45% and 5.28%±0.30%, respectively. The pH sensitivity was confirmed by changes in particle size, critical micelle concentration, and transmittance as a function of pH. MTT assay showed that PHIS/FA/PM had higher cytotoxicity at pH 6.0 than at pH 7.4, and lower cytotoxicity in the presence of free FA. Confocal laser scanning microscope images demonstrated a time-dependent and FA-inhibited cellular uptake. In vivo imaging confirmed that the mixed micelles targeted accumulation at tumor sites and the tumor inhibition rate was 85.97%. The results proved that the mixed micelle system fabricated by MPEG-PHIS and FA-PEG-VE is a promising approach to improve antitumor efficacy.

pH-sensitive and folic acid-targeted MPEG-PHIS/FA-PEG-VE mixed micelles for the delivery of PTX-VE and their antitumor activity

PubMed Central

Di, Yan; Li, Ting; Zhu, Zhihong; Chen, Fen; Jia, Lianqun; Liu, Wenbing; Gai, Xiumei; Wang, Yingying; Pan, Weisan; Yang, Xinggang

2017-01-01

The aim of this study was to simultaneously introduce pH sensitivity and folic acid (FA) targeting into a micelle system to achieve quick drug release and to enhance its accumulation in tumor cells. Paclitaxel-(+)-α-tocopherol (PTX-VE)-loaded mixed micelles (PHIS/FA/PM) fabricated by poly(ethylene glycol) methyl ether-poly(histidine) (MPEG-PHIS) and folic acid-poly(ethylene glycol)-(+)-α-tocopherol (FA-PEG-VE) were characterized by dynamic light scattering and transmission electron microscopy (TEM). The mixed micelles had a spherical morphology with an average diameter of 137.0±6.70 nm and a zeta potential of −48.7±4.25 mV. The drug encapsulation and loading efficiencies were 91.06%±2.45% and 5.28%±0.30%, respectively. The pH sensitivity was confirmed by changes in particle size, critical micelle concentration, and transmittance as a function of pH. MTT assay showed that PHIS/FA/PM had higher cytotoxicity at pH 6.0 than at pH 7.4, and lower cytotoxicity in the presence of free FA. Confocal laser scanning microscope images demonstrated a time-dependent and FA-inhibited cellular uptake. In vivo imaging confirmed that the mixed micelles targeted accumulation at tumor sites and the tumor inhibition rate was 85.97%. The results proved that the mixed micelle system fabricated by MPEG-PHIS and FA-PEG-VE is a promising approach to improve antitumor efficacy. PMID:28860753

Fusaric Acid Induces DNA Damage and Post-Translational Modifications of p53 in Human Hepatocellular Carcinoma (HepG2 ) Cells.

PubMed

Ghazi, Terisha; Nagiah, Savania; Tiloke, Charlette; Sheik Abdul, Naeem; Chuturgoon, Anil A

2017-11-01

Fusaric acid (FA), a common fungal contaminant of maize, is known to mediate toxicity in plants and animals; however, its mechanism of action is unclear. p53 is a tumor suppressor protein that is activated in response to cellular stress. The function of p53 is regulated by post-translational modifications-ubiquitination, phosphorylation, and acetylation. This study investigated a possible mechanism of FA induced toxicity in the human hepatocellular carcinoma (HepG 2 ) cell line. The effect of FA on DNA integrity and post-translational modifications of p53 were investigated. Methods included: (a) culture and treatment of HepG 2 cells with FA (IC 50 : 580.32 μM, 24 h); (b) comet assay (DNA damage); (c) Western blots (protein expression of p53, MDM2, p-Ser-15-p53, a-K382-p53, a-CBP (K1535)/p300 (K1499), HDAC1 and p-Ser-47-Sirt1); and (d) Hoechst 33342 assay (apoptosis analysis). FA caused DNA damage in HepG 2 cells relative to the control (P < 0.0001). FA decreased the protein expression of p53 (0.24-fold, P = 0.0004) and increased the expression of p-Ser-15-p53 (12.74-fold, P = 0.0126) and a-K382-p53 (2.24-fold, P = 0.0096). This occurred despite the significant decrease in the histone acetyltransferase, a-CBP (K1535)/p300 (K1499) (0.42-fold, P = 0.0023) and increase in the histone deacetylase, p-Ser-47-Sirt1 (1.22-fold, P = 0.0020). The expression of MDM2, a negative regulator of p53, was elevated in the FA treatment compared to the control (1.83-fold, P < 0.0001). FA also inhibited cell proliferation and induced apoptosis in HepG 2 cells as evidenced by the Hoechst assay. Together, these results indicate that FA is genotoxic and post-translationally modified p53 leading to HepG 2 cell death. J. Cell. Biochem. 118: 3866-3874, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Identification and validation of FaP1D7, a putative marker associated with the biosynthesis of methyl butanoate in cultivated strawberry (Fragaria x ananassa).

PubMed

Gor, Mian Chee; Candappa, Chrishani; de Silva, Thishakya; Mantri, Nitin; Pang, Edwin

2017-12-12

Breeding strawberry (Fragaria x ananassa) with enhanced fruit flavour is one of the top breeding goals of many strawberry-producing countries. Although several genes involved in the biosynthetic pathways of key aroma compounds have been identified, the development and application of molecular markers associated with fruit flavour remain limited. This study aims to identify molecular markers closely linked to genes controlling strawberry aroma. A purpose-built Subtracted Diversity Array (SDA) known as Fragaria Discovery Panel (FDP) was used for marker screening. Polymorphic sequences associated with key aroma compounds were identified from two DNA bulks with extreme phenotypes, established using 50 F 1 progeny plants derived from Juliette X 07-102-41 cross, two strawberry genotypes differing in aroma profile. A total of 49 polymorphic markers for eight key aroma compounds were detected using genotypic data of the extreme DNA bulks and phenotypic data obtained from gas chromatography-mass spectrometry (GC-MS). A similarity search against the physical maps of Fragaria vesca revealed that FaP1D7 is linked to genes potentially involved in the synthesis of methyl butanoate. A C/T SNP was detected within the feature, which could possibly be converted to a molecular tool for rapid screening of the strawberry accessions for their methyl butanoate production capacity.

The B-cell translocation gene 1 (CgBTG1) identified in oyster Crassostrea gigas exhibit multiple functions in immune response.

PubMed

Liu, Rui; Cheng, Qi; Wang, Xiudan; Chen, Hao; Wang, Weilin; Zhang, Huan; Wang, Lingling; Song, Linsheng

2017-02-01

B-cell translocation gene 1 (BTG1) is a member of the anti-proliferative gene family, which plays important roles in regulation of cell cycle. In the present study, a B-cell translocation gene 1 molecule homologue (designed CgBTG1) are identified and characterized in oyster Crassostrea gigas. CgBTG1 contains a conserved BTG domain with Box A and Box B motifs, and it shares high similarities with both BTG1 and BTG2 proteins in vertebrates. CgBTG1 mRNA is predominantly expressed in hemocytes, and its expression level in hemocytes is significantly up-regulated at 6 h (5.40-fold, p < 0.01) post Vibrio splendidus stimulation. The apoptosis rate of oyster hemocytes is significantly decreased (p < 0.05) after CgBTG1 interfered by dsRNA (dsCgBTG1). This is indicated that CgBTG1 participated in the regulation of oyster hemocytes apoptosis. Furthermore, CgBTG1 could also induce the apoptosis of cancer cells (HeLa, A549 and BEL7402) in vitro. Compared with normal oysters, both vessel-like structures and muscle fibers in CgBTG1 interfered oysters are severely damaged after V. splendidus challenge in paraffin section, considering that CgBTG1 possessed an analogous feature of angiogenesis for maintenance of vessel-like structures in adductor muscle of oyster. The results suggests that CgBTG1 is a multi-functional molecule involved in the immune response of C. gigas against pathogen infection, which provides more clues for intensive studies of BTG family proteins in invertebrates. Copyright © 2016 Elsevier Ltd. All rights reserved.

Defective homing is associated with altered Cdc42 activity in cells from patients with Fanconi anemia group A

PubMed Central

Zhang, Xiaoling; Shang, Xun; Guo, Fukun; Murphy, Kim; Kirby, Michelle; Kelly, Patrick; Reeves, Lilith; Smith, Franklin O.; Williams, David A.

2008-01-01

Previous studies showed that Fanconi anemia (FA) murine stem cells have defective reconstitution after bone marrow (BM) transplantation. The mechanism underlying this defect is not known. Here, we report defective homing of FA patient BM progenitors transplanted into mouse models. Using cells from patients carrying mutations in FA complementation group A (FA-A), we show that when transplanted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) recipient mice, FA-A BM cells exhibited impaired homing activity. FA-A cells also showed defects in both cell-cell and cell-matrix adhesion. Complementation of FA-A deficiency by reexpression of FANCA readily restored adhesion of FA-A cells. A significant decrease in the activity of the Rho GTPase Cdc42 was found associated with these defective functions in patient-derived cells, and expression of a constitutively active Cdc42 mutant was able to rescue the adhesion defect of FA-A cells. These results provide the first evidence that FA proteins influence human BM progenitor homing and adhesion via the small GTPase Cdc42-regulated signaling pathway. PMID:18565850

Microarray Analysis of LTR Retrotransposon Silencing Identifies Hdac1 as a Regulator of Retrotransposon Expression in Mouse Embryonic Stem Cells

PubMed Central

Madej, Monika J.; Taggart, Mary; Gautier, Philippe; Garcia-Perez, Jose Luis; Meehan, Richard R.; Adams, Ian R.

2012-01-01

Retrotransposons are highly prevalent in mammalian genomes due to their ability to amplify in pluripotent cells or developing germ cells. Host mechanisms that silence retrotransposons in germ cells and pluripotent cells are important for limiting the accumulation of the repetitive elements in the genome during evolution. However, although silencing of selected individual retrotransposons can be relatively well-studied, many mammalian retrotransposons are seldom analysed and their silencing in germ cells, pluripotent cells or somatic cells remains poorly understood. Here we show, and experimentally verify, that cryptic repetitive element probes present in Illumina and Affymetrix gene expression microarray platforms can accurately and sensitively monitor repetitive element expression data. This computational approach to genome-wide retrotransposon expression has allowed us to identify the histone deacetylase Hdac1 as a component of the retrotransposon silencing machinery in mouse embryonic stem cells, and to determine the retrotransposon targets of Hdac1 in these cells. We also identify retrotransposons that are targets of other retrotransposon silencing mechanisms such as DNA methylation, Eset-mediated histone modification, and Ring1B/Eed-containing polycomb repressive complexes in mouse embryonic stem cells. Furthermore, our computational analysis of retrotransposon silencing suggests that multiple silencing mechanisms are independently targeted to retrotransposons in embryonic stem cells, that different genomic copies of the same retrotransposon can be differentially sensitive to these silencing mechanisms, and helps define retrotransposon sequence elements that are targeted by silencing machineries. Thus repeat annotation of gene expression microarray data suggests that a complex interplay between silencing mechanisms represses retrotransposon loci in germ cells and embryonic stem cells. PMID:22570599

RfpA, RfpB, and RfpC are the master control elements of far-red light photoacclimation (FaRLiP)

DOE PAGES

Zhao, Chi; Gan, Fei; Shen, Gaozhong; ...

2015-11-25

Terrestrial cyanobacteria often occur in niches tha tare strongly enriched in far-redlight (FRL; λ > 700nm). Some cyanobacteria exhibit a complex and extensive photoacclimation response, known as FRLphotoacclimation(FaRLiP).During the FaRLiP response, specialized paralogous proteins replace 17 core subunits of the three major photosynthetic complexes: Photosystem (PS)I, PSII,and the phycobilisome. Additionally, the cells synthesize both chlorophyll (Chl) f and Chl d.Using biparental mating from Escherichia coli, we constructed null mutants of three genes, rfpA, rfpB,and rfpC, in the cyanobacteria Chlorogloeopsis fritschii PCC 9212 and Chroococcidiopsis thermalis PCC 7203.The resulting mutants were no longer able to modify their photosynthetic apparatus to absorbmore » FRL, were no longer able to synthesize Chl f, in appropriately synthesized Chl d in white light,and were unable to transcribe genes of the FaRLiP gene cluster. We conclude that RfpA, RfpB, and RfpC constitute a FRL-activated signal transduction cascade that is the master control switch for the FaRLiP response. FRL is proposed to activate (or inactivate) the histidine kinase activity of RfpA, which leads to formation of the active state of RfpB, the key response regulator and transcription activator. RfpC may act as a phosphate shuttle between RfpA and RfpB. Our results show that reverse genetics via conjugation will be a powerful approach in detailed studies of the FaRLiP response.« less

RfpA, RfpB, and RfpC are the master control elements of far-red light photoacclimation (FaRLiP)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Chi; Gan, Fei; Shen, Gaozhong

Terrestrial cyanobacteria often occur in niches tha tare strongly enriched in far-redlight (FRL; λ > 700nm). Some cyanobacteria exhibit a complex and extensive photoacclimation response, known as FRLphotoacclimation(FaRLiP).During the FaRLiP response, specialized paralogous proteins replace 17 core subunits of the three major photosynthetic complexes: Photosystem (PS)I, PSII,and the phycobilisome. Additionally, the cells synthesize both chlorophyll (Chl) f and Chl d.Using biparental mating from Escherichia coli, we constructed null mutants of three genes, rfpA, rfpB,and rfpC, in the cyanobacteria Chlorogloeopsis fritschii PCC 9212 and Chroococcidiopsis thermalis PCC 7203.The resulting mutants were no longer able to modify their photosynthetic apparatus to absorbmore » FRL, were no longer able to synthesize Chl f, in appropriately synthesized Chl d in white light,and were unable to transcribe genes of the FaRLiP gene cluster. We conclude that RfpA, RfpB, and RfpC constitute a FRL-activated signal transduction cascade that is the master control switch for the FaRLiP response. FRL is proposed to activate (or inactivate) the histidine kinase activity of RfpA, which leads to formation of the active state of RfpB, the key response regulator and transcription activator. RfpC may act as a phosphate shuttle between RfpA and RfpB. Our results show that reverse genetics via conjugation will be a powerful approach in detailed studies of the FaRLiP response.« less

Standing out from the crowd: How to identify plasma cells.

PubMed

Tellier, Julie; Nutt, Stephen L

2017-08-01

Being the sole source of antibody, plasmablasts and plasma cells are essential for protective immunity. Due to their relative rarity, heterogeneity and the loss of many canonical B-cell markers, antibody-secreting cells (ASCs) have often been problematic to identify and further characterize. In the mouse, the combination of the expression of CD138 and BLIMP-1, has led to many insights into ASC biology, although this approach requires the use of a GFP reporter strain. In the current issue of the European Journal of Immunology, two independent studies by Wilmore et al. and Pracht et al. provide alternative approaches to identify all murine ASCs using antibodies against the cell surface proteins, Sca-1 and TACI, respectively. Here we will discuss the advantages of these new approaches to identify ASCs in the context of our emerging knowledge of the cell surface phenotype and gene expression program of various ASC subsets in the murine and human systems. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Continuous time Bayesian networks identify Prdm1 as a negative regulator of TH17 cell differentiation in humans

PubMed Central

Acerbi, Enzo; Viganò, Elena; Poidinger, Michael; Mortellaro, Alessandra; Zelante, Teresa; Stella, Fabio

2016-01-01

T helper 17 (TH17) cells represent a pivotal adaptive cell subset involved in multiple immune disorders in mammalian species. Deciphering the molecular interactions regulating TH17 cell differentiation is particularly critical for novel drug target discovery designed to control maladaptive inflammatory conditions. Using continuous time Bayesian networks over a time-course gene expression dataset, we inferred the global regulatory network controlling TH17 differentiation. From the network, we identified the Prdm1 gene encoding the B lymphocyte-induced maturation protein 1 as a crucial negative regulator of human TH17 cell differentiation. The results have been validated by perturbing Prdm1 expression on freshly isolated CD4+ naïve T cells: reduction of Prdm1 expression leads to augmentation of IL-17 release. These data unravel a possible novel target to control TH17 polarization in inflammatory disorders. Furthermore, this study represents the first in vitro validation of continuous time Bayesian networks as gene network reconstruction method and as hypothesis generation tool for wet-lab biological experiments. PMID:26976045

The Suppressive Activity of Fucofuroeckol-A Derived from Brown Algal Ecklonia stolonifera Okamura on UVB-Induced Mast Cell Degranulation

PubMed Central

Vo, Thanh Sang; Kim, Se-Kwon; Ngo, Dai Hung; Yoon, Na-Young; Bach, Long Giang; Hang, Nguyen Thi Nhat; Ngo, Dai Nghiep

2018-01-01

UV light, especially UVB, is known as a trigger of allergic reaction, leading to mast cell degranulation and histamine release. In this study, phlorotannin Fucofuroeckol-A (F-A) derived from brown algal Ecklonia stolonifera Okamura was evaluated for its protective capability against UVB-induced allergic reaction in RBL-2H3 mast cells. It was revealed that F-A significantly suppress mast cell degranulation via decreasing histamine release as well as intracellular Ca2+ elevation at the concentration of 50 μM. Moreover, the inhibitory effect of F-A on IL-1β and TNF-α productions was also evidenced. Notably, the protective activity of F-A against mast cell degranulation was found due to scavenging ROS production. Accordingly, F-A from brown algal E. stolonifera was suggested to be promising candidate for its protective capability against UVB-induced allergic reaction. PMID:29300311

Flow cytometric characterization of the response of Fanconi's anemia cells to mitomycin C treatment.

PubMed

Kaiser, T N; Lojewski, A; Dougherty, C; Juergens, L; Sahar, E; Latt, S A

1982-03-01

DNA flow histogram analysis, using 33342 Hoechst as a stain, has been used to detect the effect of the potentially bifunctional alkylating agent, mitomycin C (MMC) on dermal fibroblasts from patients with Fanconi's anemia (FA), a hereditary human disease characterized by pancytopenia, hypersensitivity to DNA-crosslinking agents, congenital abnormalities and a predisposition for neoplasia. At 24 or 48 hr after a 2-hr exposure to 0.05 or 0.10 micrograms/ml MMC, (3)HdT incorporation was reduced to a greater extent in FA cells than in normal cells. Cells sorted from the last half of S phase showed a slightly greater inhibition of (3)HdT incorporation than did those sorted from the first half of S. Fanconi's anemia cells exhibited a marked accumulation in the G(2) + M peak of flow histograms following exposure to MMC. Twenty-four hr after treatment with .0.5 micrograms/ml MMC, the G(2) + M fraction of FA cells (eight lines) increased to more than 0.5 from a control value of approximately 0.02. Both normals (six lines) and heterozygotes (eight lines) showed, on the average, much less of a G(2) + M increment than did FA cells, even after exposure to 0.1 micrograms/ml MMC. Examination of cells sorted from the G(2) + M peak revealed that MMC-treated FA cells were blocked prior to mitosis. To determine whether the response of FA cells was specific for bifunctional alkylating agent, cells were also treated with ethylmethanesulfonate, a monofunctional agent. Twenty-four hours after exposure to 0.25 or 0.5 mg/ml ethylmethanesulfonate, FA and normal cells showed similar, small increases in the G(2) + M peak. The results suggest the utility of flow cytometry in the diagnostic evaluation of fibroblasts from patients suspected of having Fanconi's anemia.

Production Support Flight Control Computers: Research Capability for F/A-18 Aircraft at Dryden Flight Research Center

NASA Technical Reports Server (NTRS)

Carter, John F.

1997-01-01

NASA Dryden Flight Research Center (DFRC) is working with the United States Navy to complete ground testing and initiate flight testing of a modified set of F/A-18 flight control computers. The Production Support Flight Control Computers (PSFCC) can give any fleet F/A-18 airplane an in-flight, pilot-selectable research control law capability. NASA DFRC can efficiently flight test the PSFCC for the following four reasons: (1) Six F/A-18 chase aircraft are available which could be used with the PSFCC; (2) An F/A-18 processor-in-the-loop simulation exists for validation testing; (3) The expertise has been developed in programming the research processor in the PSFCC; and (4) A well-defined process has been established for clearing flight control research projects for flight. This report presents a functional description of the PSFCC. Descriptions of the NASA DFRC facilities, PSFCC verification and validation process, and planned PSFCC projects are also provided.

Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control

PubMed Central

Grant, Gavin D.; Gamsby, Joshua; Martyanov, Viktor; Brooks, Lionel; George, Lacy K.; Mahoney, J. Matthew; Loros, Jennifer J.; Dunlap, Jay C.; Whitfield, Michael L.

2012-01-01

We developed a system to monitor periodic luciferase activity from cell cycle–regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle–regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle–dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant. PMID:22740631

Identification and quantification of fumonisin A1, A2, and A3 in corn by high-resolution liquid chromatography-orbitrap mass spectrometry.

PubMed

Tamura, Masayoshi; Mochizuki, Naoki; Nagatomi, Yasushi; Harayama, Koichi; Toriba, Akira; Hayakawa, Kazuichi

2015-02-16

Three compounds, hypothesized as fumonisin A1 (FA1), fumonisin A2 (FA2), and fumonisin A3 (FA3), were detected in a corn sample contaminated with mycotoxins by high-resolution liquid chromatography-Orbitrap mass spectrometry (LC-Orbitrap MS). One of them has been identified as FA1 synthesized by the acetylation of fumonisin B1 (FB1), and established a method for its quantification. Herein, we identified the two remaining compounds as FA2 and FA3, which were acetylated fumonisin B2 (FB2) and fumonisin B3 (FB3), respectively. Moreover, we examined a method for the simultaneous analysis of FA1, FA2, FA3, FB1, FB2, and FB3. The corn samples were prepared by extraction using a QuEChERS kit and purification using a multifunctional cartridge. The linearity, recovery, repeatability, limit of detection, and limit of quantification of the method were >0.99, 82.9%-104.6%, 3.7%-9.5%, 0.02-0.60 μg/kg, and 0.05-1.98 μg/kg, respectively. The simultaneous analysis of the six fumonisins revealed that FA1, FA2, and FA3 were present in all corn samples contaminated with FB1, FB2, and FB3. The results suggested that corn marketed for consumption can be considered as being contaminated with both the fumonisin B-series and with fumonisin A-series. This report presents the first identification and quantification of FA1, FA2, and FA3 in corn samples.

Identification and Quantification of Fumonisin A1, A2, and A3 in Corn by High-Resolution Liquid Chromatography-Orbitrap Mass Spectrometry

PubMed Central

Tamura, Masayoshi; Mochizuki, Naoki; Nagatomi, Yasushi; Harayama, Koichi; Toriba, Akira; Hayakawa, Kazuichi

2015-01-01

Three compounds, hypothesized as fumonisin A1 (FA1), fumonisin A2 (FA2), and fumonisin A3 (FA3), were detected in a corn sample contaminated with mycotoxins by high-resolution liquid chromatography-Orbitrap mass spectrometry (LC-Orbitrap MS). One of them has been identified as FA1 synthesized by the acetylation of fumonisin B1 (FB1), and established a method for its quantification. Herein, we identified the two remaining compounds as FA2 and FA3, which were acetylated fumonisin B2 (FB2) and fumonisin B3 (FB3), respectively. Moreover, we examined a method for the simultaneous analysis of FA1, FA2, FA3, FB1, FB2, and FB3. The corn samples were prepared by extraction using a QuEChERS kit and purification using a multifunctional cartridge. The linearity, recovery, repeatability, limit of detection, and limit of quantification of the method were >0.99, 82.9%–104.6%, 3.7%–9.5%, 0.02–0.60 μg/kg, and 0.05–1.98 μg/kg, respectively. The simultaneous analysis of the six fumonisins revealed that FA1, FA2, and FA3 were present in all corn samples contaminated with FB1, FB2, and FB3. The results suggested that corn marketed for consumption can be considered as being contaminated with both the fumonisin B-series and with fumonisin A-series. This report presents the first identification and quantification of FA1, FA2, and FA3 in corn samples. PMID:25690692

Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

DOE Office of Scientific and Technical Information (OSTI.GOV)

Medina, D.; Oborn, C.J.; Li, M.L.

1987-09-01

The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appearedmore » to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.« less

SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE2.6, an Ortholog of OPEN STOMATA1, Is a Negative Regulator of Strawberry Fruit Development and Ripening1[OPEN

PubMed Central

Dang, Ruihong; Li, Jinxi; Jiang, Jinzhu; Zhang, Ning; Jia, Meiru; Wei, Lingzhi; Li, Ziqiang; Li, Bingbing; Jia, Wensuo

2015-01-01

Whereas the regulatory mechanisms that direct fruit ripening have been studied extensively, little is known about the signaling mechanisms underlying this process, especially for nonclimacteric fruits. In this study, we demonstrated that a SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE2, designated as FaSnRK2.6, is a negative regulator of fruit development and ripening in the nonclimacteric fruit strawberry (Fragaria × ananassa) and can also mediate temperature-modulated strawberry fruit ripening. FaSnRK2.6 was identified as an ortholog of OPEN STOMATA1. Levels of FaSnRK2.6 transcript rapidly decreased during strawberry fruit development and ripening. FaSnRK2.6 was found to be capable of physically interacting with strawberry ABSCISIC ACID INSENSITIVE1, a negative regulator in strawberry fruit ripening. RNA interference-induced silencing of FaSnRK2.6 significantly promoted fruit ripening. By contrast, overexpression of FaSnRK2.6 arrested fruit ripening. Strawberry fruit ripening is highly sensitive to temperature, with high temperatures promoting ripening and low temperatures delaying it. As the temperature increased, the level of FaSnRK2.6 expression declined. Furthermore, manipulating the level of FaSnRK2.6 expression altered the expression of a variety of temperature-responsive genes. Taken together, this study demonstrates that FaSnRK2.6 is a negative regulator of strawberry fruit development and ripening and, furthermore, that FaSnRK2.6 mediates temperature-modulated strawberry fruit ripening. PMID:25609556

Preparation and characterization of (-)-Epigallocatechin-3-gallate (EGCG)-loaded nanoparticles and their inhibitory effects on Human breast cancer MCF-7 cells.

PubMed

Zeng, Liang; Yan, Jingna; Luo, Liyong; Ma, Mengjun; Zhu, Huiqun

2017-03-28

We were employing nanotechnology to improve the targeting ability of (-)-Epigallocatechin-3-gallate (EGCG) towards MCF-7 cells, and two kinds of EGCG nanoparticles (FA-NPS-PEG and FA-PEG-NPS) were obtained, besides, their characteristics and effects on MCF-7 cells were studied. The results indicated that (i) both FA-NPS-PEG and FA-PEG-NPS have high stabilities; (ii) their particles sizes were 185.0 ± 13.5 nm and 142.7 ± 7.2 nm, respectively; (iii) their encapsulation efficiencies of EGCG were 90.36 ± 2.20% and 39.79 ± 7.54%, respectively. (iv) there was no cytotoxicity observed in EGCG, FA-NPS-PEG and FA-PEG-NPS toward MCF-7 cells over all concentrations (0~400 μg/mL) tested; (v) EGCG, FA-NPS-PEG and FA-PEG-NPS inhibited MCF-7 cells proliferation in dose-dependent manners, with the average IC 50 of 470.5 ± 33.0, 65.9 ± 0.4 and 66.6 ± 0.6 μg/mL; (vi) EGCG, FA-NPS-PEG and FA-PEG-NPS could modulated the expressions of several key regulatory proteins in PI3K-Akt pathway such as up-regulation of PTEN, p21 and Bax, and down-regulation of p-PDK1, p-AKT, CyclinD1 and Bcl-2, which gave an illustration about the mechanism by which EGCG nanoparticles inhibited MCF-7 cells proliferation. In this study, EGCG nanoparticles can significantly enhance the targeting ability and efficacy of EGCG, which is considered to an experimental foundation for further research on its activity, targeting ability and metabolism in vivo.

Targeted delivery of 5-fluorouracil to HT-29 cells using high efficient folic acid-conjugated nanoparticles.

PubMed

Wang, Yichao; Li, Puwang; Chen, Lijue; Gao, Weimin; Zeng, Fanbo; Kong, Ling Xue

2015-02-01

The incorporation of a high percentage of targeting molecules into drug delivery system is one of the important methods for improving efficacy of targeting therapeutic drugs to cancer cells. PLGA-based drug delivery carriers with folic acid (FA) as targeting molecule have a low targeting efficiency due to a low FA conjugation ratio. In this work, we fabricated a FA-conjugated PLGA system using a crosslinker 1, 3-diaminopropane and have achieved a high conjugation ratio of 46.7% (mol/mol). The as-prepared PLGA-based biomaterial was used to encapsulate therapeutic drug 5-fluorouracil (5-FU) into nanoparticles. In the in vitro experiments, an IC₅₀ of 5.69 µg/mL has been achieved for 5-FU loaded PLGA-1, 3-diaminopropane-folic acid nanoparticles on HT-29 cancer cells and is significantly lower than that of 5-FU and 5-FU loaded PLGA nanoparticles which only have an IC₅₀ of 22.9 and 14.17 µg/mL, respectively. The fluorescent microscopy images showed that nanoparticles with FA are largely taken up by HT-29 cancer cells and the targeting nanoparticles have more affinity to cancer cells than the pure drugs and untreated nanoparticles. Therefore, the 1, 3-diaminopropane can facilitate the conjugation of FA to PLGA to form a novel polymer and 5-FU loaded PLGA-1, 3-diaminopropane-folic acid nanoparticles can be a highly efficient system for specific delivery of drugs to cancer cells.

Parallel RNAi screens across different cell lines identify generic and cell type-specific regulators of actin organization and cell morphology.

PubMed

Liu, Tao; Sims, David; Baum, Buzz

2009-01-01

In recent years RNAi screening has proven a powerful tool for dissecting gene functions in animal cells in culture. However, to date, most RNAi screens have been performed in a single cell line, and results then extrapolated across cell types and systems. Here, to dissect generic and cell type-specific mechanisms underlying cell morphology, we have performed identical kinome RNAi screens in six different Drosophila cell lines, derived from two distinct tissues of origin. This analysis identified a core set of kinases required for normal cell morphology in all lines tested, together with a number of kinases with cell type-specific functions. Most significantly, the screen identified a role for minibrain (mnb/DYRK1A), a kinase associated with Down's syndrome, in the regulation of actin-based protrusions in CNS-derived cell lines. This cell type-specific requirement was not due to the peculiarities in the morphology of CNS-derived cells and could not be attributed to differences in mnb expression. Instead, it likely reflects differences in gene expression that constitute the cell type-specific functional context in which mnb/DYRK1A acts. Using parallel RNAi screens and gene expression analyses across cell types we have identified generic and cell type-specific regulators of cell morphology, which include mnb/DYRK1A in the regulation of protrusion morphology in CNS-derived cell lines. This analysis reveals the importance of using different cell types to gain a thorough understanding of gene function across the genome and, in the case of kinases, the difficulties of using the differential gene expression to predict function.

Biallelic inactivation of REV7 is associated with Fanconi anemia.

PubMed

Bluteau, Dominique; Masliah-Planchon, Julien; Clairmont, Connor; Rousseau, Alix; Ceccaldi, Raphael; Dubois d'Enghien, Catherine; Bluteau, Olivier; Cuccuini, Wendy; Gachet, Stéphanie; Peffault de Latour, Régis; Leblanc, Thierry; Socié, Gérard; Baruchel, André; Stoppa-Lyonnet, Dominique; D'Andrea, Alan D; Soulier, Jean

2016-09-01

Fanconi anemia (FA) is a recessive genetic disease characterized by congenital abnormalities, chromosome instability, progressive bone marrow failure (BMF), and a strong predisposition to cancer. Twenty FA genes have been identified, and the FANC proteins they encode cooperate in a common pathway that regulates DNA crosslink repair and replication fork stability. We identified a child with severe BMF who harbored biallelic inactivating mutations of the translesion DNA synthesis (TLS) gene REV7 (also known as MAD2L2), which encodes the mutant REV7 protein REV7-V85E. Patient-derived cells demonstrated an extended FA phenotype, which included increased chromosome breaks and G2/M accumulation upon exposure to DNA crosslinking agents, γH2AX and 53BP1 foci accumulation, and enhanced p53/p21 activation relative to cells derived from healthy patients. Expression of WT REV7 restored normal cellular and functional phenotypes in the patient's cells, and CRISPR/Cas9 inactivation of REV7 in a non-FA human cell line produced an FA phenotype. Finally, silencing Rev7 in primary hematopoietic cells impaired progenitor function, suggesting that the DNA repair defect underlies the development of BMF in FA. Taken together, our genetic and functional analyses identified REV7 as a previously undescribed FA gene, which we term FANCV.

Biallelic inactivation of REV7 is associated with Fanconi anemia

PubMed Central

Masliah-Planchon, Julien; Clairmont, Connor; Rousseau, Alix; Ceccaldi, Raphael; Dubois d’Enghien, Catherine; Bluteau, Olivier; Cuccuini, Wendy; Gachet, Stéphanie; Peffault de Latour, Régis; Leblanc, Thierry; Socié, Gérard; Baruchel, André; Stoppa-Lyonnet, Dominique; D’Andrea, Alan D.

2016-01-01

Fanconi anemia (FA) is a recessive genetic disease characterized by congenital abnormalities, chromosome instability, progressive bone marrow failure (BMF), and a strong predisposition to cancer. Twenty FA genes have been identified, and the FANC proteins they encode cooperate in a common pathway that regulates DNA crosslink repair and replication fork stability. We identified a child with severe BMF who harbored biallelic inactivating mutations of the translesion DNA synthesis (TLS) gene REV7 (also known as MAD2L2), which encodes the mutant REV7 protein REV7-V85E. Patient-derived cells demonstrated an extended FA phenotype, which included increased chromosome breaks and G2/M accumulation upon exposure to DNA crosslinking agents, γH2AX and 53BP1 foci accumulation, and enhanced p53/p21 activation relative to cells derived from healthy patients. Expression of WT REV7 restored normal cellular and functional phenotypes in the patient’s cells, and CRISPR/Cas9 inactivation of REV7 in a non-FA human cell line produced an FA phenotype. Finally, silencing Rev7 in primary hematopoietic cells impaired progenitor function, suggesting that the DNA repair defect underlies the development of BMF in FA. Taken together, our genetic and functional analyses identified REV7 as a previously undescribed FA gene, which we term FANCV. PMID:27500492

RfpA, RfpB, and RfpC are the Master Control Elements of Far-Red Light Photoacclimation (FaRLiP)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Chi; Gan, Fei; Shen, Gaozhong

Terrestrial cyanobacteria often occur in niches that are strongly enriched in far-red light (FRL; λ > 700 nm). Some cyanobacteria exhibit a complex and extensive photoacclimation response, known as FRL photoacclimation (FaRLiP). During the FaRLiP response, specialized paralogous proteins replace 17 core subunits of the three major photosynthetic complexes: Photosystem (PS) I, PS II, and the phycobilisome. Additionally, the cells synthesize both chlorophyll (Chl) f and Chl d. Using biparental mating from Escherichia coli, we constructed null mutants of three genes, rfpA, rfpB, and rfpC, in the cyanobacteria Chlorogloeopsis fritschii PCC 9212 and Chroococcidiopsis thermalis PCC 7203. The resulting mutantsmore » were no longer able to modify their photosynthetic apparatus to absorb FRL, were no longer able to synthesize Chl f, inappropriately synthesized Chl d in white light, and were unable to transcribe genes of the FaRLiP gene cluster. We conclude that RfpA, RfpB, and RfpC constitute a FRL-activated signal transduction cascade that is the master control switch for the FaRLiP response. FRL is proposed to activate (or inactivate) the histidine kinase activity of RfpA, which leads to formation of the active state of RfpB, the key response regulator and transcription activator. RfpC may act as a phosphate shuttle between RfpA and RfpB. Our results show that reverse genetics via conjugation will be a powerful approach in detailed studies of the FaRLiP response.« less

Targeting PYK2 mediates microenvironment-specific cell death in multiple myeloma

PubMed Central

Meads, MB; Fang, B; Mathews, L; Gemmer, J; Nong, L; Rosado-Lopez, I; Nguyen, T; Ring, JE; Matsui, W; MacLeod, AR; Pachter, JA; Hazlehurst, LA; Koomen, JM; Shain, KH

2015-01-01

Multiple myeloma (MM) remains an incurable malignancy due, in part, to the influence of the bone marrow microenvironment on survival and drug response. Identification of microenvironment-specific survival signaling determinants is critical for the rational design of therapy and elimination of MM. Previously, we have shown that collaborative signaling between β1 integrin-mediated adhesion to fibronectin and interleukin-6 confers a more malignant phenotype via amplification of signal transducer and activator of transcription 3 (STAT3) activation. Further characterization of the events modulated under these conditions with quantitative phosphotyrosine profiling identified 193 differentially phosphorylated peptides. Seventy-seven phosphorylations were upregulated upon adhesion, including PYK2/FAK2, Paxillin, CASL and p130CAS consistent with focal adhesion (FA) formation. We hypothesized that the collaborative signaling between β1 integrin and gp130 (IL-6 beta receptor, IL-6 signal transducer) was mediated by FA formation and proline-rich tyrosine kinase 2 (PYK2) activity. Both pharmacological and molecular targeting of PYK2 attenuated the amplification of STAT3 phosphorylation under co-stimulatory conditions. Co-culture of MM cells with patient bone marrow stromal cells (BMSC) showed similar β1 integrin-specific enhancement of PYK2 and STAT3 signaling. Molecular and pharmacological targeting of PYK2 specifically induced cell death and reduced clonogenic growth in BMSC-adherent myeloma cell lines, aldehyde dehydrogenase-positive MM cancer stem cells and patient specimens. Finally, PYK2 inhibition similarly attenuated MM progression in vivo. These data identify a novel PYK2-mediated survival pathway in MM cells and MM cancer stem cells within the context of microenvironmental cues, providing preclinical support for the use of the clinical stage FAK/PYK2 inhibitors for treatment of MM, especially in a minimal residual disease setting. PMID:26387544

Profiling human breast epithelial cells using single cell RNA sequencing identifies cell diversity.

PubMed

Nguyen, Quy H; Pervolarakis, Nicholas; Blake, Kerrigan; Ma, Dennis; Davis, Ryan Tevia; James, Nathan; Phung, Anh T; Willey, Elizabeth; Kumar, Raj; Jabart, Eric; Driver, Ian; Rock, Jason; Goga, Andrei; Khan, Seema A; Lawson, Devon A; Werb, Zena; Kessenbrock, Kai

2018-05-23

Breast cancer arises from breast epithelial cells that acquire genetic alterations leading to subsequent loss of tissue homeostasis. Several distinct epithelial subpopulations have been proposed, but complete understanding of the spectrum of heterogeneity and differentiation hierarchy in the human breast remains elusive. Here, we use single-cell mRNA sequencing (scRNAseq) to profile the transcriptomes of 25,790 primary human breast epithelial cells isolated from reduction mammoplasties of seven individuals. Unbiased clustering analysis reveals the existence of three distinct epithelial cell populations, one basal and two luminal cell types, which we identify as secretory L1- and hormone-responsive L2-type cells. Pseudotemporal reconstruction of differentiation trajectories produces one continuous lineage hierarchy that closely connects the basal lineage to the two differentiated luminal branches. Our comprehensive cell atlas provides insights into the cellular blueprint of the human breast epithelium and will form the foundation to understand how the system goes awry during breast cancer.

FA composition of heart and skeletal muscle during embryonic development of the king penguin.

PubMed

Decrock, Frederic; Groscolas, Rene; Speake, Brian K

2002-04-01

Since the yolk lipids of the king penguin (Aptenodytes patagonicus) naturally contain the highest concentrations of DHA and EPA yet reported for the eggs of any avian species, the effects of this (n-3)-rich yolk on the FA profiles of the embryonic heart and skeletal muscle were investigated. The concentrations (mg/g wet tissue) of phospholipid (PL) in the developing heart and leg muscle of the penguin doubled between days 27 and 55 from the beginning of egg incubation (i.e., from the halfway stage of embryonic development to 2 d posthatch), whereas no net increase occurred in pectoral muscle. During this period, the concentration of TAG in heart decreased by half but increased two- and sixfold in leg and pectoral muscle, respectively. The most notable change in cholesteryl ester concentration occurred in pectoral muscle, increasing ninefold between days 27 and 55. Arachidonic acid (ARA) was the major polyunsaturate in PL of the penguin's heart, where it formed about 20% (w/w) of FA at day 55. At the equivalent developmental stage, the heart PL of the chicken contained a 1.3-fold greater proportion of ARA, contained a fifth less DHA, and was almost devoid of EPA, whereas the latter FA was a significant component (7% of FA) of penguin heart PL. Similarly, in PL of leg and pectoral muscle, the chicken displayed about 1.4-fold more ARA, up to 50% less DHA, and far less EPA in comparison with the penguin. Thus, although ARA-rich PL profiles are achieved in the heart and muscle of the penguin embryo, these profiles are significantly affected by the high n-3 content of the yolk.

A turn-on fluorescent probe for endogenous formaldehyde in the endoplasmic reticulum of living cells

NASA Astrophysics Data System (ADS)

Tang, Yonghe; Ma, Yanyan; Xu, An; Xu, Gaoping; Lin, Weiying

2017-06-01

As the simplest aldehyde compounds, formaldehyde (FA) is implicated in nervous system diseases and cancer. Endoplasmic reticulum is an organelle that plays important functions in living cells. Accordingly, the development of efficient methods for FA detection in the endoplasmic reticulum (ER) is of great biomedical importance. In this work, we developed the first ER-targeted fluorescent FA probe Na-FA-ER. The detection is based on the condensation reaction of the hydrazine group and FA to suppress the photo-induced electron transfer (PET) pathway, resulting in a fluorescence increase. The novel Na-FA-ER showed high sensitivity to FA. In addition, the Na-FA-ER enabled the bio-imaging of exogenous and endogenous FA in living HeLa cells. Most significantly, the new Na-FA-ER was employed to visualize the endogenous FA in the ER in living cells for the first time.

Birth order and recalled childhood gender nonconformity in Samoan men and fa'afafine.

PubMed

Semenyna, Scott W; VanderLaan, Doug P; Vasey, Paul L

2017-04-01

Having a greater than average number of older biological brothers is a robust correlate of male androphilia (i.e., sexual attraction and arousal to adult males). Previous investigations have sought to understand whether this fraternal birth order (FBO) effect is also systematically related to recalled indicators of childhood gender nonconformity (CGN). However, these investigations have relied on data from low-fertility Western populations in which expressions of femininity in male children are routinely stigmatized and consequently, suppressed. The present study examined the FBO effect (among other sibship characteristics) and recalled indicators of CGN in Samoa, a high-fertility population, whose members are relatively tolerant of male femininity. Indeed, Samoans identify feminine androphilic males as belonging to an alternative gender category, known locally as fa'afafine. The present study compared the sibship characteristics of 231 fa'afafine and 231 opposite-sex attracted men from Samoa, as well as how these characteristics related to recalled CGN. Results replicated the well-established FBO effect for predicting male sexual orientation, with each older brother increasing the odds of being androphilic by 21%. However, no relationship was found between the number of older brothers (or other siblings) a participant had and their recalled CGN. Although fa'afafine reported significantly more CGN than Samoan men, CGN did not mediate the FBO effect, nor did the FBO effect and CGN interact to predict male sexual orientation. These findings are consistent with previous studies suggesting that the FBO effect is associated with male sexual orientation, but not childhood female-typical gender expression among androphilic males. © 2017 Wiley Periodicals, Inc.

Preparation and characterization of (−)-Epigallocatechin-3-gallate (EGCG)-loaded nanoparticles and their inhibitory effects on Human breast cancer MCF-7 cells

PubMed Central

Zeng, Liang; Yan, Jingna; Luo, Liyong; Ma, Mengjun; Zhu, Huiqun

2017-01-01

We were employing nanotechnology to improve the targeting ability of (−)-Epigallocatechin-3-gallate (EGCG) towards MCF-7 cells, and two kinds of EGCG nanoparticles (FA-NPS-PEG and FA-PEG-NPS) were obtained, besides, their characteristics and effects on MCF-7 cells were studied. The results indicated that (i) both FA-NPS-PEG and FA-PEG-NPS have high stabilities; (ii) their particles sizes were 185.0 ± 13.5 nm and 142.7 ± 7.2 nm, respectively; (iii) their encapsulation efficiencies of EGCG were 90.36 ± 2.20% and 39.79 ± 7.54%, respectively. (iv) there was no cytotoxicity observed in EGCG, FA-NPS-PEG and FA-PEG-NPS toward MCF-7 cells over all concentrations (0~400 μg/mL) tested; (v) EGCG, FA-NPS-PEG and FA-PEG-NPS inhibited MCF-7 cells proliferation in dose-dependent manners, with the average IC50 of 470.5 ± 33.0, 65.9 ± 0.4 and 66.6 ± 0.6 μg/mL; (vi) EGCG, FA-NPS-PEG and FA-PEG-NPS could modulated the expressions of several key regulatory proteins in PI3K-Akt pathway such as up-regulation of PTEN, p21 and Bax, and down-regulation of p-PDK1, p-AKT, CyclinD1 and Bcl-2, which gave an illustration about the mechanism by which EGCG nanoparticles inhibited MCF-7 cells proliferation. In this study, EGCG nanoparticles can significantly enhance the targeting ability and efficacy of EGCG, which is considered to an experimental foundation for further research on its activity, targeting ability and metabolism in vivo. PMID:28349962

Architectural Kansei of ‘Wall’ in The Façade Design by Le Corbusier

NASA Astrophysics Data System (ADS)

Sendai, Shoichiro

The purpose of this paper is to discuss the modern architect Le Corbusier's architectural Kansei (sensibility) on wall in site environment through the analysis of his façade design, using Œuvres complètes (1910-1965, 8 vols., Les éditions d'architecture, Artemis, Zurich) and Le Corbusier Archives (1982-1984, 32 vols., Garland Publishing, Inc. and Fondation Le Corbusier, New York, London, Paris). At first, I arrange five façade types, according to the explanation by Le Corbusier ; ‘fenêtre en longueur (strip window)’, ‘pan de verre (glass wall)’, ‘brise-soleil (sun-breaker)’, ‘loggia’ and ‘claustra’. Through the analysis of the relationship between these types and the design process of each building, we find that Le Corbusier's façade design includes the affirmation and the negation of the ‘wall’ at the same time. In fact, the nature of façade modification during design process is divers: increase in transparency, decrease in transparency and spatialization of façade. That means, Le Corbusier studied the environmental condition by these façade types, and tried to realize the phenomenal openness. This trial bases on the function of architectural Kansei as correspondence between body and environment beyond the physical design.

A Novel Single-Cell FISH-Flow Assay Identifies Effector Memory CD4+ T cells as a Major Niche for HIV-1 Transcription in HIV-Infected Patients.

PubMed

Grau-Expósito, Judith; Serra-Peinado, Carla; Miguel, Lucia; Navarro, Jordi; Curran, Adrià; Burgos, Joaquin; Ocaña, Imma; Ribera, Esteban; Torrella, Ariadna; Planas, Bibiana; Badía, Rosa; Castellví, Josep; Falcó, Vicenç; Crespo, Manuel; Buzon, Maria J

2017-07-11

Cells that actively transcribe HIV-1 have been defined as the "active viral reservoir" in HIV-infected individuals. However, important technical limitations have precluded the characterization of this specific viral reservoir during both treated and untreated HIV-1 infections. Here, we used a novel single-cell RNA fluorescence in situ hybridization-flow cytometry (FISH-flow) assay that requires only 15 million unfractionated peripheral blood mononuclear cells (PBMCs) to characterize the specific cell subpopulations that transcribe HIV RNA in different subsets of CD4 + T cells. In samples from treated and untreated HIV-infected patients, effector memory CD4 + T cells were the main cell population supporting HIV RNA transcription. The number of cells expressing HIV correlated with the plasma viral load, intracellular HIV RNA, and proviral DNA quantified by conventional methods and inversely correlated with the CD4 + T cell count and the CD4/CD8 ratio. We also found that after ex vivo infection of unstimulated PBMCs, HIV-infected T cells upregulated the expression of CD32. In addition, this new methodology detected increased numbers of primary cells expressing viral transcripts and proteins after ex vivo viral reactivation with latency reversal agents. This RNA FISH-flow technique allows the identification of the specific cell subpopulations that support viral transcription in HIV-1-infected individuals and has the potential to provide important information on the mechanisms of viral pathogenesis, HIV persistence, and viral reactivation. IMPORTANCE Persons infected with HIV-1 contain several cellular viral reservoirs that preclude the complete eradication of the viral infection. Using a novel methodology, we identified effector memory CD4 + T cells, immune cells preferentially located in inflamed tissues with potent activity against pathogens, as the main cells encompassing the transcriptionally active HIV-1 reservoir in patients on antiretroviral therapy. Importantly

Live-cell imaging RNAi screen identifies PP2A–B55α and importin-β1 as key mitotic exit regulators in human cells

PubMed Central

Schmitz, Michael H. A.; Held, Michael; Janssens, Veerle; Hutchins, James R. A.; Hudecz, Otto; Ivanova, Elitsa; Goris, Jozef; Trinkle-Mulcahy, Laura; Lamond, Angus I.; Poser, Ina; Hyman, Anthony A.; Mechtler, Karl; Peters, Jan-Michael; Gerlich, Daniel W.

2013-01-01

When vertebrate cells exit mitosis various cellular structures are re-organized to build functional interphase cells1. This depends on Cdk1 (cyclin dependent kinase 1) inactivation and subsequent dephosphorylation of its substrates2–4. Members of the protein phosphatase 1 and 2A (PP1 and PP2A) families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit5,6, but how this relates to postmitotic reassembly of interphase structures in intact cells is not known. Here, we use a live-cell imaging assay and RNAi knockdown to screen a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identify a trimeric PP2A–B55α complex as a key factor in mitotic spindle breakdown and postmitotic reassembly of the nuclear envelope, Golgi apparatus and decondensed chromatin. Using a chemically induced mitotic exit assay, we find that PP2A–B55α functions downstream of Cdk1 inactivation. PP2A–B55α isolated from mitotic cells had reduced phosphatase activity towards the Cdk1 substrate, histone H1, and was hyper-phosphorylated on all subunits. Mitotic PP2A complexes co-purified with the nuclear transport factor importin-β1, and RNAi depletion of importin-β1 delayed mitotic exit synergistically with PP2A–B55α. This demonstrates that PP2A–B55α and importin-β1 cooperate in the regulation of postmitotic assembly mechanisms in human cells. PMID:20711181

Total Risk Integrated Methodology (TRIM) - TRIM.FaTE

EPA Pesticide Factsheets

TRIM.FaTE is a spatially explicit, compartmental mass balance model that describes the movement and transformation of pollutants over time, through a user-defined, bounded system that includes both biotic and abiotic compartments.

Dynamic behavior of reactive aluminum nanoparticle-fluorinated acrylic (AlFA) polymer composites

NASA Astrophysics Data System (ADS)

Crouse, Christopher A.; White, Brad; Spowart, Jonathan E.

2011-06-01

The dynamic behavior of aluminum nanoparticle-fluorinated acrylic (AlFA) composite materials has been explored under high strain rates. Cylindrical pellets of the AlFA composite materials were mounted onto copper sabots and impacted against a rigid anvil at velocities between 100 and 400 m/s utilizing a Taylor gas gun apparatus to achieve strain rates on the order of 104 /s. A framing camera was used to record the compaction and reaction events that occurred upon contact of the pellet with the anvil. Under both open air and vacuum environments the AlFA composites demonstrated high reactivity suggesting that the particles are primarily reacting with the fluorinated matrix. We hypothesize, based upon the compaction history of these materials, that reaction is initiated when the oxide shells on the aluminum nanoparticles are broken due an interparticle contact deformation process. We have investigated this hypothesis through altering the particle loading in the AlFA composites as well as impact velocities. This data and the corresponding trends will be presented in detail.

Chalcones suppress fatty acid-induced lipid accumulation through a LKB1/AMPK signaling pathway in HepG2 cells.

PubMed

Zhang, Tianshun; Yamamoto, Norio; Ashida, Hitoshi

2014-06-01

Excessive lipid accumulation in the liver has been proposed to cause hyperlipidemia, diabetes and fatty liver disease. 4-Hydroxyderricin (4HD), xanthoangelol (XAG), cardamonin (CAR) and flavokawain B (FKB) are chalcones that have exhibited various biological effects against obesity, inflammation, and diabetes; however, little is known about the inhibitory effects of these chalcones on fatty liver disease. In the present study, we investigated the ability of 4HD, XAG, CAR, and FKB to reduce lipid accumulation in hepatocytes. When HepG2 cells were treated with a mixture of fatty acids (FAs; palmitic acid : oleic acid = 1 : 2 ratio), significant lipid accumulation was observed. Under the same experimental conditions, addition of chalcones at 5 μM significantly suppressed the FA-induced lipid accumulation. We found that the expression of sterol regulatory element-binding protein-1 (SREBP-1), a key molecule involved in lipogenesis, was decreased in these chalcone-treated cells. We also found that these chalcones increased the expression of peroxisome proliferator-activated receptor α (PPARα), which is involved in FA oxidation. Moreover, these chalcones increased phosphorylation of AMP-activated protein kinase (AMPK) and liver kinase B1 (LKB1), upstream regulators of SREBP-1 and PPARα. We confirmed that an AMPK inhibitor, compound C, reversed chalcone-induced changes in SREBP-1 and PPARα expression in the HepG2 cells. Collectively, we found that 4HD, XAG, CAR, and XAG attenuated lipid accumulation through activation of the LKB1/AMPK signaling pathway in HepG2 cells.

AluY-mediated germline deletion, duplication and somatic stem cell reversion in UBE2T defines a new subtype of Fanconi anemia.

PubMed

Virts, Elizabeth L; Jankowska, Anna; Mackay, Craig; Glaas, Marcel F; Wiek, Constanze; Kelich, Stephanie L; Lottmann, Nadine; Kennedy, Felicia M; Marchal, Christophe; Lehnert, Erik; Scharf, Rüdiger E; Dufour, Carlo; Lanciotti, Marina; Farruggia, Piero; Santoro, Alessandra; Savasan, Süreyya; Scheckenbach, Kathrin; Schipper, Jörg; Wagenmann, Martin; Lewis, Todd; Leffak, Michael; Farlow, Janice L; Foroud, Tatiana M; Honisch, Ellen; Niederacher, Dieter; Chakraborty, Sujata C; Vance, Gail H; Pruss, Dmitry; Timms, Kirsten M; Lanchbury, Jerry S; Alpi, Arno F; Hanenberg, Helmut

2015-09-15

Fanconi anemia (FA) is a rare inherited disorder clinically characterized by congenital malformations, progressive bone marrow failure and cancer susceptibility. At the cellular level, FA is associated with hypersensitivity to DNA-crosslinking genotoxins. Eight of 17 known FA genes assemble the FA E3 ligase complex, which catalyzes monoubiquitination of FANCD2 and is essential for replicative DNA crosslink repair. Here, we identify the first FA patient with biallelic germline mutations in the ubiquitin E2 conjugase UBE2T. Both mutations were aluY-mediated: a paternal deletion and maternal duplication of exons 2-6. These loss-of-function mutations in UBE2T induced a cellular phenotype similar to biallelic defects in early FA genes with the absence of FANCD2 monoubiquitination. The maternal duplication produced a mutant mRNA that could encode a functional protein but was degraded by nonsense-mediated mRNA decay. In the patient's hematopoietic stem cells, the maternal allele with the duplication of exons 2-6 spontaneously reverted to a wild-type allele by monoallelic recombination at the duplicated aluY repeat, thereby preventing bone marrow failure. Analysis of germline DNA of 814 normal individuals and 850 breast cancer patients for deletion or duplication of UBE2T exons 2-6 identified the deletion in only two controls, suggesting aluY-mediated recombinations within the UBE2T locus are rare and not associated with an increased breast cancer risk. Finally, a loss-of-function germline mutation in UBE2T was detected in a high-risk breast cancer patient with wild-type BRCA1/2. Cumulatively, we identified UBE2T as a bona fide FA gene (FANCT) that also may be a rare cancer susceptibility gene. © The Author 2015. Published by Oxford University Press.

Effector T cells require fatty acid metabolism during murine graft-versus-host disease

PubMed Central

Byersdorfer, Craig A.; Tkachev, Victor; Opipari, Anthony W.; Goodell, Stefanie; Swanson, Jacob; Sandquist, Stacy; Glick, Gary D.; Ferrara, James L. M.

2013-01-01

Activated T cells require increased energy to proliferate and mediate effector functions, but the metabolic changes that occur in T cells following stimulation in vivo are poorly understood, particularly in the context of inflammation. We have previously shown that T cells activated during graft-versus-host disease (GVHD) primarily rely on oxidative phosphorylation to synthesize adenosine 5′-triphosphate. Here, we demonstrate that alloreactive effector T cells (Teff) use fatty acids (FAs) as a fuel source to support their in vivo activation. Alloreactive T cells increased FA transport, elevated levels of FA oxidation enzymes, up-regulated transcriptional coactivators to drive oxidative metabolism, and increased their rates of FA oxidation. Importantly, increases in FA transport and up-regulation of FA oxidation machinery occurred specifically in T cells during GVHD and were not seen in Teff following acute activation. Pharmacological blockade of FA oxidation decreased the survival of alloreactive T cells but did not influence the survival of T cells during normal immune reconstitution. These studies suggest that pathways controlling FA metabolism might serve as therapeutic targets to treat GVHD and other T-cell–mediated immune diseases. PMID:24046012

"What D'ya Mean, Project SOL-FA?"

ERIC Educational Resources Information Center

Hardman, Olga S.

1981-01-01

Since 1977, Project Sol-fa, funded with an ESEA Title IV-C grant has provided Harrison County primary teachers with inservice training in the Kodaly method of music education. This article provides information on program funding, costs, and accomplishments. Evaluation forms and the inservice syllabus are appended. (SJL)

Somatic mosaicism in Fanconi anemia: Evidence of genotypic reversion in lymphohematopoietic stem cells

PubMed Central

Gregory, John J.; Wagner, John E.; Verlander, Peter C.; Levran, Orna; Batish, Sat Dev; Eide, Cindy R.; Steffenhagen, Amy; Hirsch, Betsy; Auerbach, Arleen D.

2001-01-01

Somatic mosaicism has been observed previously in the lymphocyte population of patients with Fanconi anemia (FA). To identify the cellular origin of the genotypic reversion, we examined each lymphohematopoietic and stromal cell lineage in an FA patient with a 2815–2816ins19 mutation in FANCA and known lymphocyte somatic mosaicism. DNA extracted from individually plucked peripheral blood T cell colonies and marrow colony-forming unit granulocyte–macrophage and burst-forming unit erythroid cells revealed absence of the maternal FANCA exon 29 mutation in 74.0%, 80.3%, and 86.2% of colonies, respectively. These data, together with the absence of the FANCA exon 29 mutation in Epstein–Barr virus-transformed B cells and its presence in fibroblasts, indicate that genotypic reversion, most likely because of back mutation, originated in a lymphohematopoietic stem cell and not solely in a lymphocyte population. Contrary to a predicted increase in marrow cellularity resulting from reversion in a hematopoietic stem cell, pancytopenia was progressive. Additional evaluations revealed a partial deletion of 11q in 3 of 20 bone marrow metaphase cells. By using interphase fluorescence in situ hybridization with an MLL gene probe mapped to band 11q23 to identify colony-forming unit granulocyte–macrophage and burst-forming unit erythroid cells with the 11q deletion, the abnormal clone was exclusive to colonies with the FANCA exon 29 mutation. Thus, we demonstrate the spontaneous genotypic reversion in a lymphohematopoietic stem cell. The subsequent development of a clonal cytogenetic abnormality in nonrevertant cells suggests that ex vivo correction of hematopoietic stem cells by gene transfer may not be sufficient for providing life-long stable hematopoiesis in patients with FA. PMID:11226273

Leaching of biocides from façades under natural weather conditions.

PubMed

Burkhardt, M; Zuleeg, S; Vonbank, R; Bester, K; Carmeliet, J; Boller, M; Wangler, T

2012-05-15

Biocides are included in organic building façade coatings as protection against biological attack by algae and fungi but have the potential to enter the environment via leaching into runoff from wind driven rain. The following field study correlates wind driven rain to runoff and measured the release of several commonly used organic biocides (terbutryn, Irgarol 1051, diuron, isoproturon, OIT, DCOIT) in organic façade coatings from four coating systems. During one year of exposure of a west oriented model house façade in the Zurich, Switzerland area, an average of 62.7 L/m(2), or 6.3% of annual precipitation came off the four façade panels installed as runoff. The ISO method for calculating wind driven rain loads is adapted to predict runoff and can be used in the calculation of emissions in the field. Biocide concentrations tend to be higher in the early lifetime of the coatings and then reach fairly consistent levels later, generally ranging on the order of mg/L or hundreds of μg/L. On the basis of the amount remaining in the film after exposure, the occurrence of transformation products, and the calculated amounts in the leachate, degradation plays a significant role in the overall mass balance.

Role of the combination of FA and T2* parameters as a new diagnostic method in therapeutic evaluation of parkinson's disease.

PubMed

Fang, Yuan; Zheng, Tao; Liu, Lanxiang; Gao, Dawei; Shi, Qinglei; Dong, Yanchao; Du, Dan

2017-11-17

Simple diffusion delivery (SDD) has attained good effects with only tiny amounts of drugs. Fractional anisotropy (FA) and relaxation time T2* that indicate the integrity of fiber tracts and iron concentration within brain tissue were used to evaluate the therapeutic effect of SDD. To evaluate therapeutic effect of SDD in the Parkinson's disease (PD) rat model with FA and T2* parameters. Prospective case-control animal study. Thirty-two male Sprague Dawley rats (eight normal, eight PD, eight SDD, and eight subcutaneous injection rats). Single-shot spin echo echo-planar imaging and fast low-angle shot T 2 WI sequences at 3.0T. Parameters of FA and T2* on the treated side of the substantia nigra were measured to evaluate the therapeutic effect of SDD in a PD rat model. The effects of time on FA and T2* values were analyzed by repeated measurement tests. A one-way analysis of variance was conducted, followed by individual comparisons of the mean FA and T2* values at different timepoints. The FA values on the treated side of the substantia nigra in the SDD treatment group and subcutaneous injection treatment group were significantly higher at week 1 and lower at week 6 than that of the PD control group (SDD vs. PD, week 1, adjusted P = 0.012; subcutaneous vs. PD, week 1, adjusted P < 0.001; SDD vs. PD, week 6, adjusted P = 0.004; subcutaneous vs. PD, week 6, adjusted P = 0.024). The T2* parameter in the SDD treatment group and subcutaneous injection treatment group was significantly higher than that in the PD control group at week 6 (SDD vs. PD, adjusted P = 0.032; subcutaneous vs. PD, adjusted P < 0.001). The combination of FA and T2* parameters can potentially serve as a new effective evaluation method of the therapeutic effect of SDD. 1 Technical Efficacy: Stage 4 J. Magn. Reson. Imaging 2017. © 2017 International Society for Magnetic Resonance in Medicine.

DOG-1 Is the Caenorhabditis elegans BRIP1/FANCJ Homologue and Functions in Interstrand Cross-Link Repair▿

PubMed Central

Youds, Jillian L.; Barber, Louise J.; Ward, Jordan D.; Collis, Spencer J.; O'Neil, Nigel J.; Boulton, Simon J.; Rose, Ann M.

2008-01-01

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by defective DNA interstrand cross-link (ICL) repair. Here, we show that DOG-1 is the Caenorhabditis elegans homologue of FANCJ, a helicase mutated in FA-J patients. DOG-1 performs a conserved role in ICL repair, as dog-1 mutants are hypersensitive to ICL-inducing agents, but not to UVC irradiation or X rays. Genetic analysis indicated that dog-1 is epistatic with fcd-2 (C. elegans FANCD2) but is nonepistatic with brc-1 (C. elegans BRCA1), thus establishing the existence of two distinct pathways of ICL repair in worms. Furthermore, DOG-1 is dispensable for FCD-2 and RAD-51 focus formation, suggesting that DOG-1 operates downstream of FCD-2 and RAD-51 in ICL repair. DOG-1 was previously implicated in poly(G)/poly(C) (G/C) tract maintenance during DNA replication. G/C tracts remain stable in the absence of ATL-1, CLK-2 (FA pathway activators), FCD-2, BRC-2, and MLH-1 (associated FA components), implying that DOG-1 is the sole FA component required for G/C tract maintenance in a wild-type background. However, FCD-2 is required to promote deletion-free repair at G/C tracts in dog-1 mutants, consistent with a role for FA factors at the replication fork. The functional conservation between DOG-1 and FANCJ suggests a possible role for FANCJ in G/C tract maintenance in human cells. PMID:18086896

Molecular regulation and physiological functions of a novel FaHsfA2c cloned from tall fescue conferring plant tolerance to heat stress.

PubMed

Wang, Xiuyun; Huang, Wanlu; Liu, Jun; Yang, Zhimin; Huang, Bingru

2017-02-01

Heat stress transcription factors (HSFs) compose a large gene family, and different members play differential roles in regulating plant responses to abiotic stress. The objectives of this study were to identify and characterize an A2-type HSF, FaHsfA2c, in a cool-season perennial grass tall fescue (Festuca arundinacea Schreb.) for its association with heat tolerance and to determine the underlying physiological functions and regulatory mechanisms of FaHsfA2c imparting plant tolerance to heat stress. FaHsfA2c was localized in nucleus and exhibited a rapid transcriptional increase in leaves and roots during early phase of heat stress. Ectopic expression of FaHsfA2c improved basal and acquired thermotolerance in wild-type Arabidopsis and also restored heat-sensitive deficiency of hsfa2 mutant. Overexpression of FaHsfA2c in tall fescue enhanced plant tolerance to heat by triggering transcriptional regulation of heat-protective gene expression, improving photosynthetic capacity and maintaining plant growth under heat stress. Our results indicated that FaHsfA2c acted as a positive regulator conferring thermotolerance improvement in Arabidopsis and tall fescue, and it could be potentially used as a candidate gene for genetic modification and molecular breeding to develop heat-tolerant cool-season grass species. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Prismatic louver active façades for natural illumination and thermal energy gain in high-rise and commercial buildings

NASA Astrophysics Data System (ADS)

Vlachokostas, A.; Volkmann, C.; Madamopoulos, N.

2013-06-01

their surroundings, the proposed multi-purpose prismatic louver façade can be responsive and resilient to daytime sky conditions, environmental temperatures and occupant needs by exploiting options presented by the three sides of the prismatic louvers. The façade is highly configurable since every side of the prismatic louver façade can perform a different operation. The prism itself operates as a redirector of sunlight from the glass façade to the ceiling and consequently diffuses the sunlight throughout the room, providing higher and more uniform illumination levels. In addition, each side of the prismatic louver can be implemented in multiple ways (e.g., visibly transparent photovoltaic cells, luminescent solar concentrators). The ability to rotate the prismatic louvers along their axes allows the user to expose a set of different surfaces to the sun's radiation in accordance with different climatic conditions and occupant needs. Thus, the prismatic louvers help achieve a selective control and management of the incoming light that allows us to manipulate the incoming energy for the benefit of the building and its occupants.

Integrative kinome profiling identifies mTORC1/2 inhibition as treatment strategy in ovarian clear cell carcinoma.

PubMed

Caumanns, Joseph J; Berns, Katrien; Wisman, G Bea A; Fehrmann, Rudolf S N; Tomar, Tushar; Klip, Harry; Meersma, Gert Jan; Hijmans, E Marielle; Gennissen, Annemiek; Duiker, Evelien W; Weening, Desiree; Itamochi, Hiroaki; Kluin, Roelof Jc; Reyners, An K L; Birrer, Michael J; Salvesen, Helga B; Vergote, Ignace; Van Nieuwenhuysen, Els; Brenton, James D; Braicu, Elena I; Kupryjanczyk, Jolanta; Spiewankiewicz, Beata; Mittempergher, Lorenza; Bernards, Rene; van der Zee, Ate G J; de Jong, Steven

2018-04-23

Advanced stage ovarian clear cell carcinoma (OCCC) is unresponsive to conventional platinum-based chemotherapy. Frequent alterations in OCCC include deleterious mutations in the tumor suppressor ARID1A and activating mutations in the PI3K subunit PIK3CA. In this study, we aimed to identify currently unknown mutated kinases in OCCC patients and test druggability of downstream affected pathways in OCCC models. In a large set of OCCC patients (n=124), the human kinome (518 kinases) and additional cancer related genes were sequenced and copy number alterations were determined. Genetically characterized OCCC cell lines (n=17) and OCCC patient-derived xenografts (n=3) were used for drug testing of ERBB tyrosine kinase inhibitors erlotinib and lapatinib, the PARP inhibitor olaparib and the mTORC1/2 inhibitor AZD8055. We identified several putative driver mutations in kinases at low frequency that were not previously annotated in OCCC. Combining mutations and copy number alterations, 91% of all tumors are affected in the PI3K/AKT/mTOR pathway, the MAPK pathway or the ERBB family of receptor tyrosine kinases and 82% in the DNA repair pathway. Strong p-S6 staining in OCCC patients suggests high mTORC1/2 activity. We consistently found that the majority of OCCC cell lines are especially sensitive to mTORC1/2 inhibition by AZD8055 and not towards drugs targeting ERBB family of receptor tyrosine kinases or DNA repair signaling. We subsequently demonstrated the efficacy of mTORC1/2 inhibition in all our unique OCCC patient-derived xenograft models. These results propose mTORC1/2 inhibition as an effective treatment strategy in OCCC. Copyright ©2018, American Association for Cancer Research.

Optical characterization and bandgap engineering of flat and wrinkle-textured FA0.83Cs0.17Pb(I1-xBrx)3 perovskite thin films

NASA Astrophysics Data System (ADS)

Tejada, A.; Braunger, S.; Korte, L.; Albrecht, S.; Rech, B.; Guerra, J. A.

2018-05-01

The complex refractive indices of formamidinium cesium lead mixed-halide [FA0.83Cs0.17Pb(I1- xBrx)3] perovskite thin films of compositions ranging from x = 0 to 0.4, with both flat and wrinkle-textured surface topographies, are reported. The films are characterized using a combination of variable angle spectroscopic ellipsometry and spectral transmittance in the wavelength range of 190 nm to 850 nm. Optical constants, film thicknesses and roughness layers are obtained point-by-point by minimizing a global error function, without using optical dispersion models, and including topographical information supplied by a laser confocal microscope. To evaluate the bandgap engineering potential of the material, the optical bandgaps and Urbach energies are then accurately determined by applying a band fluctuation model for direct semiconductors, which considers both the Urbach tail and the fundamental band-to-band absorption region in a single equation. With this information, the composition yielding the optimum bandgap of 1.75 eV for a Si-perovskite tandem solar cell is determined.

Disrupted Signaling through the Fanconi Anemia Pathway Leads to Dysfunctional Hematopoietic Stem Cell Biology: Underlying Mechanisms and Potential Therapeutic Strategies

PubMed Central

Geiselhart, Anja; Lier, Amelie; Walter, Dagmar; Milsom, Michael D.

2012-01-01

Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. FA patients suffer to varying degrees from a heterogeneous range of developmental defects and, in addition, have an increased likelihood of developing cancer. Almost all FA patients develop a severe, progressive bone marrow failure syndrome, which impacts upon the production of all hematopoietic lineages and, hence, is thought to be driven by a defect at the level of the hematopoietic stem cell (HSC). This hypothesis would also correlate with the very high incidence of MDS and AML that is observed in FA patients. In this paper, we discuss the evidence that supports the role of dysfunctional HSC biology in driving the etiology of the disease. Furthermore, we consider the different model systems currently available to study the biology of cells defective in the FA signaling pathway and how they are informative in terms of identifying the physiologic mediators of HSC depletion and dissecting their putative mechanism of action. Finally, we ask whether the insights gained using such disease models can be translated into potential novel therapeutic strategies for the treatment of the hematologic disorders in FA patients. PMID:22675615

Systems biology approach identifies the kinase Csnk1a1 as a regulator of the DNA damage response in embryonic stem cells.

PubMed

Carreras Puigvert, Jordi; von Stechow, Louise; Siddappa, Ramakrishnaiah; Pines, Alex; Bahjat, Mahnoush; Haazen, Lizette C J M; Olsen, Jesper V; Vrieling, Harry; Meerman, John H N; Mullenders, Leon H F; van de Water, Bob; Danen, Erik H J

2013-01-22

In pluripotent stem cells, DNA damage triggers loss of pluripotency and apoptosis as a safeguard to exclude damaged DNA from the lineage. An intricate DNA damage response (DDR) signaling network ensures that the response is proportional to the severity of the damage. We combined an RNA interference screen targeting all kinases, phosphatases, and transcription factors with global transcriptomics and phosphoproteomics to map the DDR in mouse embryonic stem cells treated with the DNA cross-linker cisplatin. Networks derived from canonical pathways shared in all three data sets were implicated in DNA damage repair, cell cycle and survival, and differentiation. Experimental probing of these networks identified a mode of DNA damage-induced Wnt signaling that limited apoptosis. Silencing or deleting the p53 gene demonstrated that genotoxic stress elicited Wnt signaling in a p53-independent manner. Instead, this response occurred through reduced abundance of Csnk1a1 (CK1α), a kinase that inhibits β-catenin. Together, our findings reveal a balance between p53-mediated elimination of stem cells (through loss of pluripotency and apoptosis) and Wnt signaling that attenuates this response to tune the outcome of the DDR.

Folic acid functionalized surface highlights 5-methylcytosine-genomic content within circulating tumor cells.

PubMed

Malara, Natalia; Coluccio, Maria Laura; Limongi, Tania; Asande, Monica; Trunzo, Valentina; Cojoc, Gheorghe; Raso, Cinzia; Candeloro, Patrizio; Perozziello, Gerardo; Raimondo, Raffaella; De Vitis, Stefania; Roveda, Laura; Renne, Maria; Prati, Ubaldo; Mollace, Vincenzo; Di Fabrizio, Enzo

2014-11-12

Although the detection of methylated cell free DNA represents one of the most promising approaches for relapse risk assessment in cancer patients, the low concentration of cell-free circulating DNA constitutes the biggest obstacle in the development of DNA methylation-based biomarkers from blood. This paper describes a method for the measurement of genomic methylation content directly on circulating tumor cells (CTC), which could be used to deceive the aforementioned problem. Since CTC are disease related blood-based biomarkers, they result essential to monitor tumor's stadiation, therapy, and early relapsing lesions. Within surface's bio-functionalization and cell's isolation procedure standardization, the presented approach reveals a singular ability to detect high 5-methylcytosine CTC-subset content in the whole CTC compound, by choosing folic acid (FA) as transducer molecule. Sensitivity and specificity, calculated for FA functionalized surface (FA-surface), result respectively on about 83% and 60%. FA-surface, allowing the detection and characterization of early metastatic dissemination, provides a unique advance in the comprehension of tumors progression and dissemination confirming the presence of CTC and its association with high risk of relapse. This functionalized surface identifying and quantifying high 5-methylcytosine CTC-subset content into the patient's blood lead significant progress in cancer risk assessment, also providing a novel therapeutic strategy. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fanconi's Anemia Effect or Sickle Cell Anemia Effect: That is the Question.

PubMed

Unal, Sule; Chui, David H K; Gumruk, Fatma

2015-01-01

A 16-year-old boy who was diagnosed to have sickle cell anemia was referred to our center. The parental consanguinity, growth retardation and dysmorphic features prompted a search for possible Fanconi's Anemia (FA). The diepoxybutane (DEB) test was positive, confirming FA. The interaction of both diseases might account for his relatively mild phenotype in terms of both sickle cell anemia (or Hb S, HBB: c.20A > T) and FA. The high Hb F level that might be related to concomitant FA, may have caused a milder phenotype of sickle cell anemia, whereas nitric oxide (NO) depletion as a consequence of sickle cell anemia, may have caused a delay in the bone marrow failure of FA.

Identifying new lignin bioengineering targets: 1. Monolignol-substitute impacts on lignin formation and cell wall fermentability

PubMed Central

2010-01-01

Background Recent discoveries highlighting the metabolic malleability of plant lignification indicate that lignin can be engineered to dramatically alter its composition and properties. Current plant biotechnology efforts are primarily aimed at manipulating the biosynthesis of normal monolignols, but in the future apoplastic targeting of phenolics from other metabolic pathways may provide new approaches for designing lignins that are less inhibitory toward the enzymatic hydrolysis of structural polysaccharides, both with and without biomass pretreatment. To identify promising new avenues for lignin bioengineering, we artificially lignified cell walls from maize cell suspensions with various combinations of normal monolignols (coniferyl and sinapyl alcohols) plus a variety of phenolic monolignol substitutes. Cell walls were then incubated in vitro with anaerobic rumen microflora to assess the potential impact of lignin modifications on the enzymatic degradability of fibrous crops used for ruminant livestock or biofuel production. Results In the absence of anatomical constraints to digestion, lignification with normal monolignols hindered both the rate and extent of cell wall hydrolysis by rumen microflora. Inclusion of methyl caffeate, caffeoylquinic acid, or feruloylquinic acid with monolignols considerably depressed lignin formation and strikingly improved the degradability of cell walls. In contrast, dihydroconiferyl alcohol, guaiacyl glycerol, epicatechin, epigallocatechin, and epigallocatechin gallate readily formed copolymer-lignins with normal monolignols; cell wall degradability was moderately enhanced by greater hydroxylation or 1,2,3-triol functionality. Mono- or diferuloyl esters with various aliphatic or polyol groups readily copolymerized with monolignols, but in some cases they accelerated inactivation of wall-bound peroxidase and reduced lignification; cell wall degradability was influenced by lignin content and the degree of ester group hydroxylation

Quantifying folic acid-functionalized multi-walled carbon nanotubes bound to colorectal cancer cells for improved photothermal ablation

NASA Astrophysics Data System (ADS)

Graham, Elizabeth G.; MacNeill, Christopher M.; Levi-Polyachenko, Nicole H.

2013-05-01

Peritoneal metastases of colorectal cancer are a significant challenge in the field of medicine today due to poor results of systemic chemotherapy caused by the poor diffusion of drugs across the blood-peritoneal barrier. Multi-walled carbon nanotubes (MWNTs) are a biocompatible nanomaterial that strongly absorb near-infrared light to locally heat the surrounding area. Colorectal cancer is known to overexpress folate receptor; therefore, folic acid (FA) was covalently attached to MWNTs to target colorectal cancer cells. Results from real-time polymerase chain reaction found differing expression of folate receptor-α in two colorectal cancer cell lines, RKO and HCT116, as well as a healthy epithelial cell line, HEPM. A spectrophotometric method was developed to quantify the mass of MWNTs bound to cells, and it was determined that FA-targeted MWNTs resulted in a 400-500 % greater affinity for colorectal cancer cells than untargeted MWNTs. The non-cancerous cell line, HEPM, had higher non-specific MWNT interaction and similar MWNT-FA affinity. Stimulated by 1,064 nm light, FA-functionalized MWNTs caused a 50-60 % decrease in colorectal cancer cell viability compared to a 4-10 % decrease caused by untargeted MWNTs. Our results indicate that FA-targeted MWNTs may increase the therapeutic index of MWNT-induced photothermal therapy.

The role of FaBG3 in fruit ripening and B. cinerea fungal infection of strawberry.

PubMed

Li, Qian; Ji, Kai; Sun, Yufei; Luo, Hao; Wang, Hongqing; Leng, Ping

2013-10-01

In plants, β-glucosidases (BG) have been implicated in developmental and pathogen defense, and are thought to take part in abscisic acid (ABA) synthesis via hydrolysis of ABA glucose ester to release active ABA; however, there is no genetic evidence for the role of BG genes in ripening and biotic/abiotic stress in fruits. To clarify the role of BG genes in fruit, eight Fa/FvBG genes encoding β-glucosidase were isolated using information from the GenBank strawberry nucleotide database. Of the Fa/FvBG genes examined, expression of FaBG3 was the highest, showing peaks at the mature stage, coincident with the changes observed in ABA content. To verify the role of this gene, we suppressed the expression of FaBG3 via inoculation with Agrobacterium tumefaciens containing tobacco rattle virus carrying a FaBG3 fragment (RNAi). The expression of FaBG3 in FaBG3-RNAi-treated fruit was markedly reduced, and the ABA content was lower than that of the control. FaBG3-RNAi-treated fruit did not exhibit full ripening, and were firmer, had lower sugar content, and were pale compared with the control due to down-regulation of ripening-related genes. FaBG3-RNAi-treated fruit with reduced ABA levels were much more resistant to Botrytis cinerea fungus but were more sensitive to dehydration stress than control fruit. These results indicate that FaBG3 may play key roles in fruit ripening, dehydration stress and B. cinerea fungal infection in strawberries via modulation of ABA homeostasis and transcriptional regulation of ripening-related genes. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

New insights into the operative network of FaEO, an enone oxidoreductase from Fragaria x ananassa Duch.

PubMed

Collu, Gabriella; Farci, Domenica; Esposito, Francesca; Pintus, Francesca; Kirkpatrick, Joanna; Piano, Dario

2017-05-01

The 2-methylene-furan-3-one reductase or Fragaria x ananassa Enone Oxidoreductase (FaEO) catalyses the last reductive step in the biosynthesis of 4-hydroxy-2,5-dimethyl-3(2H)-furanone, a major component in the characteristic flavour of strawberries. In the present work, we describe the association between FaEO and the vacuolar membrane of strawberry fruits. Even if FaEO lacks epitopes for stable or transient membrane-interactions, it contains a calmodulin-binding region, suggesting that in vivo FaEO may be associated with the membrane via a peripheral protein complex with calmodulin. Moreover, we also found that FaEO occurs in dimeric form in vivo and, as frequently observed for calmodulin-regulated proteins, it may be expressed in different isoforms by alternative gene splicing. Further mass spectrometry analysis confirmed that the isolated FaEO consists in the already known isoform and that it is the most characteristic during ripening. Finally, a characterization by absorption spectroscopy showed that FaEO has specific flavoprotein features. The relevance of these findings and their possible physiological implications are discussed.

Folic acid-capped PEGylated magnetic nanoparticles enter cancer cells mostly via clathrin-dependent endocytosis.

PubMed

Allard-Vannier, Emilie; Hervé-Aubert, Katel; Kaaki, Karine; Blondy, Thibaut; Shebanova, Anastasia; Shaitan, Konstantin V; Ignatova, Anastasia A; Saboungi, Marie-Louise; Feofanov, Alexey V; Chourpa, Igor

2017-06-01

This work is focused on mechanisms of uptake in cancer cells of rationally designed, covalently assembled nanoparticles, made of superparamagnetic iron oxide nanoparticles (SPIONs), fluorophores (doxorubicin or Nile Blue), polyethylene glycol (PEG) and folic acid (FA), referred hereinafter as SFP-FA. SFP-FA were characterized by DLS, zetametry and fluorescence spectroscopy. The SFP-FA uptake in cancer cells was monitored using fluorescence-based methods like fluorescence-assisted cell sorting, CLSM with single-photon and two-photon excitation. The SFP-FA endocytosis was also analyzed with electron microscopy approaches: TEM, HAADF-STEM and EELS. The SFP-FA have zeta potential below -6mW and stable hydrodynamic diameter close to 100nm in aqueous suspensions of pH range from 5 to 8. They contain ca. 109 PEG-FA, 480 PEG-OCH 3 and 22-27 fluorophore molecules per SPION. The fluorophores protected under the PEG shell allows a reliable detection of intracellular NPs. SFP-FA readily enter into all the cancer cell lines studied and accumulate in lysosomes, mostly via clathrin-dependent endocytosis, whatever the FR status on the cells. The present study highlights the advantages of rational design of nanosystems as well as the possible involvement of direct molecular interactions of PEG and FA with cellular membranes, not limited to FA-FR recognition, in the mechanisms of their endocytosis. Composition, magnetic and optical properties of the SFP-FA as well their ability to enter cancer cells are promising for their applications in cancer theranosis. Combination of complementary analytical approaches is relevant to understand the nanoparticles behavior in suspension and in contact with cells. Copyright © 2016 Elsevier B.V. All rights reserved.

FaSTR DNA: a new expert system for forensic DNA analysis.

PubMed

Power, Timothy; McCabe, Brendan; Harbison, Sally Ann

2008-06-01

The automation of DNA profile analysis of reference and crime samples continues to gain pace driven in part by a realisation by the criminal justice system of the positive impact DNA technology can have in aiding in the solution of crime and the apprehension of suspects. Expert systems to automate the profile analysis component of the process are beginning to be developed. In this paper, we report the validation of a new expert system FaSTR DNA, an expert system suitable for the analysis of DNA profiles from single source reference samples and from crime samples. We compare the performance of FaSTR DNA with that of other equivalent systems, GeneMapper ID v3.2 (Applied Biosystems, Foster City, CA) and FSS-i(3) v4 (The Forensic Science Service((R)) DNA expert System Suite FSS-i(3), Forensic Science Service, Birmingham, UK) with GeneScan Analysis v3.7/Genotyper v3.7 software (Applied Biosystems, Foster City, CA, USA) with manual review. We have shown that FaSTR DNA provides an alternative solution to automating DNA profile analysis and is appropriate for implementation into forensic laboratories. The FaSTR DNA system was demonstrated to be comparable in performance to that of GeneMapper ID v3.2 and superior to that of FSS-i(3) v4 for the analysis of DNA profiles from crime samples.

A Security-façade Library for Virtual-observatory Software

NASA Astrophysics Data System (ADS)

Rixon, G.

2009-09-01

The security-façade library implements, for Java, IVOA's security standards. It supports the authentication mechanisms for SOAP and REST web-services, the sign-on mechanisms (with MyProxy, AstroGrid Accounts protocol or local credential-caches), the delegation protocol, and RFC3820-enabled HTTPS for Apache Tomcat. Using the façade, a developer who is not a security specialist can easily add access control to a virtual-observatory service and call secured services from an application. The library has been an internal part of AstroGrid software for some time and it is now offered for use by other developers.

Consecutive assessment of FA and ADC values of normal lumbar nerve roots from the junction of the dura mater.

PubMed

Miyagi, Ryo; Sakai, Toshinori; Yamabe, Eiko; Yoshioka, Hiroshi

2015-06-27

Diffusion weighted imaging (DWI) and diffusion tensor imaging (DTI) are widely used in the evaluation of the central nervous system and recently have been reported as a potential tool for diagnosis of the peripheral nerve or the lumbar nerve entrapment. The purpose of this study was to evaluate consecutive changes in apparent diffusion coefficient (ADC) and fractional anisotropy (FA) values of normal lumbar nerve roots from the junction of the dura mater. The lumbar spinal nerves were examined in 6 male healthy volunteers (mean age, 35 years) with no experiences of sciatica, with a 3.0-T MR unit using a five-element phased-array surface coil. DTI was performed with the following imaging parameters: 11084.6/73.7 ms for TR/TE; b-value, 800 s/mm2; MPG, 33 directions; slice thickness, 1.5 mm; and total scan time, 7 min 35 s. ADC and FA values at all consecutive points along the L4, L5 and S1 nerves were quantified on every 1.5 mm slice from the junction of the dura mater using short fiber tracking. ADC values of all L4, 5, and S1 nerve roots decreased linearly up to 15 mm from the dura junction and was constant distally afterward. ADC values in the proximal portion demonstrated S1 > L5 > L4 (p < 0.05). On the other hand, FA values increased linearly up to 15 mm from the dura junction, and was constant distally afterward. FA values in the proximal portion showed L4 > L5 > S1 (p < 0.05). Our study demonstrated that ADC and FA values of each L4, 5, and S1 at the proximal portion from the junction of the dura matter changed linearly. It would be useful to know the normal profile of DTI values by location of each nerve root so that we can detect subtle abnormalities in each nerve root.

Cotransport of Herbaspirillum chlorophenolicum FA1 and heavy metals in saturated porous media: column studies and modeling approaches

NASA Astrophysics Data System (ADS)

Li, X.; Xu, H.; Wu, J.

2017-12-01

For in situ biodegradation of organic contaminants in soil and groundwater, precise prediction and monitoring of the movement of the bio-agent is vital for the effectiveness of the subsurface bioremediation technologies. Therefore, the fate and transport of functional microorganisms in porous media has been extensively investigated in the literature, and the effects of a number of physical and chemical factors have been explored. During the bioremediation of contaminated sites, it is highly likely that functional bacteria and heavy metals would be simultaneously present for heavy metals often co-exist with organic contaminants like polycyclic aromatic hydrocarbons (PAHs) in polluted environment. To date, relevant studies on the interactions between heavy metals and functional agents such as PAHs-degrading bacteria are lacking and thus require investigation. In this study, the cotransport of bioremediation agents and heavy metals were evaluated through batch and column experiments. Herbaspirillum chlorophenolicum FA1, a pure bacterial strain capable of absorbing heavy metals and degrading polycyclic aromatic hydrocarbons (PAHs), was used as the model remediation agent, and metal ions of Pb(Ⅱ) and Cd(Ⅱ) were used as the representative heavy metals. Effects of metal species, the concentration of heavy metals, the sequence of entering the media, and the activity of biomass were investigated in detail. In addition, numerical simulations of breakthrough curves (BTC) data were also performed for information gathering. Results of this study could advance our understanding of interactions between functional bacteria and heavy metals during bioremediation process and help to develop successful bioremediation strategies.This work was financially supported by the National Natural Science Foundation of China -Xinjiang Project (U1503282), the National Natural Science Foundation of China (41030746, 41102148), and the Natural Science Foundation of Jiangsu Province (BK20151385

Design of MiSolFA Hard X-Ray Imager

NASA Astrophysics Data System (ADS)

Lastufka, Erica; Casadei, Diego

2017-08-01

Advances in the study of coronal electron-accelerating regions have so far been limited by the dynamic range of X-ray instruments. A quick and economical alternative to desirable focusing optics technology is stereo observation. The micro-satellite MiSolFA (Micro Solar-Flare Apparatus) is designed both as a stand-alone X-ray imaging spectrometer and a complement to the Spectrometer/Telescope for Imaging X-rays (STIX) mission. These instruments will be the first pair of cross-calibrated X-ray imaging spectrometers to look at solar flares from very different points of view. MiSolFA will achieve indirect imaging between 10 and 60 keV and provide spectroscopy up to 100 keV, equipped with grids producing moiré patterns in a similar way to STIX. New manufacturing techniques produce gold gratings on a graphite or silicon substrate, with periods ranging from 15 to 225 micrometers, separated by a distance of 15.47 cm, to achieve a spatial resolutions from 10" to 60" (as compared to RHESSI's separation of 150 cm and 1" resolution). We present the progress of the imager design, the performance of the first prototypes, and reach out to the community for further scientific objectives to consider in optimizing the final design.

Fanconi anemia protein, FANCA, associates with BRG1, a component of the human SWI/SNF complex.

PubMed

Otsuki, T; Furukawa, Y; Ikeda, K; Endo, H; Yamashita, T; Shinohara, A; Iwamatsu, A; Ozawa, K; Liu, J M

2001-11-01

Fanconi anemia (FA) is a genetic disorder that predisposes to hematopoietic failure, birth defects and cancer. We identified an interaction between the FA protein, FANCA and brm-related gene 1 (BRG1) product. BRG1 is a subunit of the SWI/SNF complex, which remodels chromatin structure through a DNA-dependent ATPase activity. FANCA was demonstrated to associate with the endogenous SWI/SNF complex. We also found a significant increase in the molecular chaperone, glucose-regulated protein 94 (GRP94) among BRG1-associated factors isolated from a FANCA-mutant cell line, which was not seen in either a normal control cell line or the mutant line complemented by wild-type FANCA. Despite this specific difference, FANCA did not appear to be absolutely required for in vitro chromatin remodeling. Finally, we demonstrated co-localization in the nucleus between transfected FANCA and BRG1. The physiological action of FANCA on the SWI/SNF complex remains to be clarified, but our work suggests that FANCA may recruit the SWI/SNF complex to target genes, thereby enabling coupled nuclear functions such as transcription and DNA repair.

Viewing time measures of sexual orientation in Samoan cisgender men who engage in sexual interactions with fa'afafine.

PubMed

Petterson, Lanna J; Dixson, Barnaby J; Little, Anthony C; Vasey, Paul L

2015-01-01

Androphilia refers to attraction to adult males, whereas gynephilia refers to attraction to adult females. The current study employed self-report and viewing time (response time latency) measures of sexual attraction to determine the sexual orientation of Samoan cisgender men (i.e., males whose gender presentation and identity is concordant with their biological sex) who engage in sexual interactions with transgender male androphiles (known locally as fa'afafine) compared to: (1) Samoan cisgender men who only engage in sexual interactions with women, and (2) fa'afafine. As expected, both measures indicated that cisgender men who only engaged in sexual interactions with women exhibited a gynephilic pattern of sexual attraction, whereas fa'afafine exhibited an androphilic one. In contrast, both measures indicated that cisgender men who engaged in sexual interactions with fa'afafine demonstrated a bisexual pattern of sexual attraction. Most of the cisgender men who exhibited bisexual viewing times did not engage in sexual activity with both men and women indicating that the manner in which bisexual patterns of sexual attraction manifest behaviorally vary from one culture to the next.

Diffusion tensor MRI tractography reveals increased fractional anisotropy (FA) in arcuate fasciculus following music-cued motor training.

PubMed

Moore, Emma; Schaefer, Rebecca S; Bastin, Mark E; Roberts, Neil; Overy, Katie

2017-08-01

Auditory cues are frequently used to support movement learning and rehabilitation, but the neural basis of this behavioural effect is not yet clear. We investigated the microstructural neuroplasticity effects of adding musical cues to a motor learning task. We hypothesised that music-cued, left-handed motor training would increase fractional anisotropy (FA) in the contralateral arcuate fasciculus, a fibre tract connecting auditory, pre-motor and motor regions. Thirty right-handed participants were assigned to a motor learning condition either with (Music Group) or without (Control Group) musical cues. Participants completed 20minutes of training three times per week over four weeks. Diffusion tensor MRI and probabilistic neighbourhood tractography identified FA, axial (AD) and radial (RD) diffusivity before and after training. Results revealed that FA increased significantly in the right arcuate fasciculus of the Music group only, as hypothesised, with trends for AD to increase and RD to decrease, a pattern of results consistent with activity-dependent increases in myelination. No significant changes were found in the left ipsilateral arcuate fasciculus of either group. This is the first evidence that adding musical cues to movement learning can induce rapid microstructural change in white matter pathways in adults, with potential implications for therapeutic clinical practice. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Mutagenesis Screen Identifies agtpbp1 and eps15L1 as Essential for T lymphocyte Development in Zebrafish.

PubMed

Seiler, Christoph; Gebhart, Nichole; Zhang, Yong; Shinton, Susan A; Li, Yue-sheng; Ross, Nicola L; Liu, Xingjun; Li, Qin; Bilbee, Alison N; Varshney, Gaurav K; LaFave, Matthew C; Burgess, Shawn M; Balciuniene, Jorune; Balciunas, Darius; Hardy, Richard R; Kappes, Dietmar J; Wiest, David L; Rhodes, Jennifer

2015-01-01

Genetic screens are a powerful tool to discover genes that are important in immune cell development and function. The evolutionarily conserved development of lymphoid cells paired with the genetic tractability of zebrafish make this a powerful model system for this purpose. We used a Tol2-based gene-breaking transposon to induce mutations in the zebrafish (Danio rerio, AB strain) genome, which served the dual purpose of fluorescently tagging cells and tissues that express the disrupted gene and provided a means of identifying the disrupted gene. We identified 12 lines in which hematopoietic tissues expressed green fluorescent protein (GFP) during embryonic development, as detected by microscopy. Subsequent analysis of young adult fish, using a novel approach in which single cell suspensions of whole fish were analyzed by flow cytometry, revealed that 8 of these lines also exhibited GFP expression in young adult cells. An additional 15 lines that did not have embryonic GFP+ hematopoietic tissue by microscopy, nevertheless exhibited GFP+ cells in young adults. RT-PCR analysis of purified GFP+ populations for expression of T and B cell-specific markers identified 18 lines in which T and/or B cells were fluorescently tagged at 6 weeks of age. As transposon insertion is expected to cause gene disruption, these lines can be used to assess the requirement for the disrupted genes in immune cell development. Focusing on the lines with embryonic GFP+ hematopoietic tissue, we identified three lines in which homozygous mutants exhibited impaired T cell development at 6 days of age. In two of the lines we identified the disrupted genes, agtpbp1 and eps15L1. Morpholino-mediated knockdown of these genes mimicked the T cell defects in the corresponding mutant embryos, demonstrating the previously unrecognized, essential roles of agtpbp1 and eps15L1 in T cell development.

Integration of ATAC-seq and RNA-seq identifies human alpha cell and beta cell signature genes.

PubMed

Ackermann, Amanda M; Wang, Zhiping; Schug, Jonathan; Naji, Ali; Kaestner, Klaus H

2016-03-01

Although glucagon-secreting α-cells and insulin-secreting β-cells have opposing functions in regulating plasma glucose levels, the two cell types share a common developmental origin and exhibit overlapping transcriptomes and epigenomes. Notably, destruction of β-cells can stimulate repopulation via transdifferentiation of α-cells, at least in mice, suggesting plasticity between these cell fates. Furthermore, dysfunction of both α- and β-cells contributes to the pathophysiology of type 1 and type 2 diabetes, and β-cell de-differentiation has been proposed to contribute to type 2 diabetes. Our objective was to delineate the molecular properties that maintain islet cell type specification yet allow for cellular plasticity. We hypothesized that correlating cell type-specific transcriptomes with an atlas of open chromatin will identify novel genes and transcriptional regulatory elements such as enhancers involved in α- and β-cell specification and plasticity. We sorted human α- and β-cells and performed the "Assay for Transposase-Accessible Chromatin with high throughput sequencing" (ATAC-seq) and mRNA-seq, followed by integrative analysis to identify cell type-selective gene regulatory regions. We identified numerous transcripts with either α-cell- or β-cell-selective expression and discovered the cell type-selective open chromatin regions that correlate with these gene activation patterns. We confirmed cell type-selective expression on the protein level for two of the top hits from our screen. The "group specific protein" (GC; or vitamin D binding protein) was restricted to α-cells, while CHODL (chondrolectin) immunoreactivity was only present in β-cells. Furthermore, α-cell- and β-cell-selective ATAC-seq peaks were identified to overlap with known binding sites for islet transcription factors, as well as with single nucleotide polymorphisms (SNPs) previously identified as risk loci for type 2 diabetes. We have determined the genetic landscape of

Flight Tests of a Ministick Controller in an F/A-18 Airplane

NASA Technical Reports Server (NTRS)

Stoliker, Patrick C.; Carter, John

2003-01-01

In March of 1999, five pilots performed flight tests to evaluate the handling qualities of an F/A-18 research airplane equipped with a small-displacement center stick (ministick) controller that had been developed for the JAS 39 Gripen airplane (a fighter/attack/ reconnaissance airplane used by the Swedish air force). For these tests, the ministick was installed in the aft cockpit (see figure) and production support flight control computers (PSFCCs) were used as interfaces between the controller hardware and the standard F/A-18 flight-control laws. The primary objective of the flight tests was to assess any changes in handling qualities of the F/A-18 airplane attributable to the mechanical characteristics of the ministick. The secondary objective was to demonstrate the capability of the PSFCCs to support flight-test experiments.

A High-Content Small Molecule Screen Identifies Sensitivity of Glioblastoma Stem Cells to Inhibition of Polo-Like Kinase 1

PubMed Central

Danovi, Davide; Folarin, Amos; Gogolok, Sabine; Ender, Christine; Elbatsh, Ahmed M. O.; Engström, Pär G.; Stricker, Stefan H.; Gagrica, Sladjana; Georgian, Ana; Yu, Ding; U, Kin Pong; Harvey, Kevin J.; Ferretti, Patrizia; Paddison, Patrick J.; Preston, Jane E.; Abbott, N. Joan; Bertone, Paul; Smith, Austin; Pollard, Steven M.

2013-01-01

Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults and there are few effective treatments. GBMs contain cells with molecular and cellular characteristics of neural stem cells that drive tumour growth. Here we compare responses of human glioblastoma-derived neural stem (GNS) cells and genetically normal neural stem (NS) cells to a panel of 160 small molecule kinase inhibitors. We used live-cell imaging and high content image analysis tools and identified JNJ-10198409 (J101) as an agent that induces mitotic arrest at prometaphase in GNS cells but not NS cells. Antibody microarrays and kinase profiling suggested that J101 responses are triggered by suppression of the active phosphorylated form of polo-like kinase 1 (Plk1) (phospho T210), with resultant spindle defects and arrest at prometaphase. We found that potent and specific Plk1 inhibitors already in clinical development (BI 2536, BI 6727 and GSK 461364) phenocopied J101 and were selective against GNS cells. Using a porcine brain endothelial cell blood-brain barrier model we also observed that these compounds exhibited greater blood-brain barrier permeability in vitro than J101. Our analysis of mouse mutant NS cells (INK4a/ARF−/−, or p53−/−), as well as the acute genetic deletion of p53 from a conditional p53 floxed NS cell line, suggests that the sensitivity of GNS cells to BI 2536 or J101 may be explained by the lack of a p53-mediated compensatory pathway. Together these data indicate that GBM stem cells are acutely susceptible to proliferative disruption by Plk1 inhibitors and that such agents may have immediate therapeutic value. PMID:24204733

Integrated proteomics identified novel activation of dynein IC2-GR-COX-1 signaling in neurofibromatosis type I (NF1) disease model cells.

PubMed

Hirayama, Mio; Kobayashi, Daiki; Mizuguchi, Souhei; Morikawa, Takashi; Nagayama, Megumi; Midorikawa, Uichi; Wilson, Masayo M; Nambu, Akiko N; Yoshizawa, Akiyasu C; Kawano, Shin; Araki, Norie

2013-05-01

Neurofibromatosis type 1 (NF1) tumor suppressor gene product, neurofibromin, functions in part as a Ras-GAP, and though its loss is implicated in the neuronal abnormality of NF1 patients, its precise cellular function remains unclear. To study the molecular mechanism of NF1 pathogenesis, we prepared NF1 gene knockdown (KD) PC12 cells, as a NF1 disease model, and analyzed their molecular (gene and protein) expression profiles with a unique integrated proteomics approach, comprising iTRAQ, 2D-DIGE, and DNA microarrays, using an integrated protein and gene expression analysis chart (iPEACH). In NF1-KD PC12 cells showing abnormal neuronal differentiation after NGF treatment, of 3198 molecules quantitatively identified and listed in iPEACH, 97 molecules continuously up- or down-regulated over time were extracted. Pathway and network analysis further revealed overrepresentation of calcium signaling and transcriptional regulation by glucocorticoid receptor (GR) in the up-regulated protein set, whereas nerve system development was overrepresented in the down-regulated protein set. The novel up-regulated network we discovered, "dynein IC2-GR-COX-1 signaling," was then examined in NF1-KD cells. Validation studies confirmed that NF1 knockdown induces altered splicing and phosphorylation patterns of dynein IC2 isomers, up-regulation and accumulation of nuclear GR, and increased COX-1 expression in NGF-treated cells. Moreover, the neurite retraction phenotype observed in NF1-KD cells was significantly recovered by knockdown of the dynein IC2-C isoform and COX-1. In addition, dynein IC2 siRNA significantly inhibited nuclear translocation and accumulation of GR and up-regulation of COX-1 expression. These results suggest that dynein IC2 up-regulates GR nuclear translocation and accumulation, and subsequently causes increased COX-1 expression, in this NF1 disease model. Our integrated proteomics strategy, which combines multiple approaches, demonstrates that NF1-related neural

Wind tunnel study of natural ventilation of building integrated photovoltaics double skin façade

NASA Astrophysics Data System (ADS)

Hudişteanu, Sebastian Valeriu; Popovici, Cătălin George; Cherecheş, Nelu-Cristian

2018-02-01

The paper presents a wind tunnel experimental analysis of a small-scale building model (1:30). The objective of the study is to determine the wind influence on the ventilation of a double skin façade channel (DSF) and the cooling effect over integrated photovoltaic panels. The tests were achieved by conceiving and implementation of an experimental program using a wind tunnel with atmospheric boundary layer. The effect of the wind over the ventilation of the horizontal channels of double skin façades is evaluated for different incident velocities. The results are generalized for the average steady state values of the velocities analysed. The experimental results put in evidence the correlation between the reference wind velocity and the dynamics of the air movement inside the double skin façade. These values are used to determine the convective heat transfer and the cooling effect of the air streams inside the channel upon the integrated photovoltaic panels. The decrease of the photovoltaic panels temperature determines a raise of 11% in efficiency and power generated.

Rad18 confers hematopoietic progenitor cell DNA damage tolerance independently of the Fanconi Anemia pathway in vivo

PubMed Central

Yang, Yang; Poe, Jonathan C.; Yang, Lisong; Fedoriw, Andrew; Desai, Siddhi; Magnuson, Terry; Li, Zhiguo; Fedoriw, Yuri; Araki, Kimi; Gao, Yanzhe; Tateishi, Satoshi; Sarantopoulos, Stefanie; Vaziri, Cyrus

2016-01-01

In cultured cancer cells the E3 ubiquitin ligase Rad18 activates Trans-Lesion Synthesis (TLS) and the Fanconi Anemia (FA) pathway. However, physiological roles of Rad18 in DNA damage tolerance and carcinogenesis are unknown and were investigated here. Primary hematopoietic stem and progenitor cells (HSPC) co-expressed RAD18 and FANCD2 proteins, potentially consistent with a role for Rad18 in FA pathway function during hematopoiesis. However, hematopoietic defects typically associated with fanc-deficiency (decreased HSPC numbers, reduced engraftment potential of HSPC, and Mitomycin C (MMC) -sensitive hematopoiesis), were absent in Rad18−/− mice. Moreover, primary Rad18−/− mouse embryonic fibroblasts (MEF) retained robust Fancd2 mono-ubiquitination following MMC treatment. Therefore, Rad18 is dispensable for FA pathway activation in untransformed cells and the Rad18 and FA pathways are separable in hematopoietic cells. In contrast with responses to crosslinking agents, Rad18−/− HSPC were sensitive to in vivo treatment with the myelosuppressive agent 7,12 Dimethylbenz[a]anthracene (DMBA). Rad18-deficient fibroblasts aberrantly accumulated DNA damage markers after DMBA treatment. Moreover, in vivo DMBA treatment led to increased incidence of B cell malignancy in Rad18−/− mice. These results identify novel hematopoietic functions for Rad18 and provide the first demonstration that Rad18 confers DNA damage tolerance and tumor-suppression in a physiological setting. PMID:26883629

A Competitive Stapled Peptide Screen Identifies a Selective Small Molecule that Overcomes MCL-1-dependent Leukemia Cell Survival

PubMed Central

Cohen, Nicole A.; Stewart, Michelle L.; Gavathiotis, Evripidis; Tepper, Jared L.; Bruekner, Susanne R.; Koss, Brian; Opferman, Joseph T.; Walensky, Loren D.

2012-01-01

SUMMARY Cancer cells hijack BCL-2 family survival proteins to suppress the death effectors and thereby enforce an immortal state. This is accomplished biochemically by an anti-apoptotic surface groove that neutralizes the pro-apoptotic BH3 α-helix of death proteins. Anti-apoptotic MCL-1 in particular has emerged as a ubiquitous resistance factor in cancer. Whereas targeting the BCL-2 anti-apoptotic subclass effectively restores the death pathway in BCL-2-dependent cancer, the development of molecules tailored to the binding specificity of MCL-1 has lagged. We previously discovered that a hydrocarbon-stapled MCL-1 BH3 helix is an exquisitely selective MCL-1 antagonist. By deploying this unique reagent in a competitive screen, we identified an MCL-1 inhibitor molecule that selectively targets the BH3-binding groove of MCL-1, neutralizes its biochemical lockhold on apoptosis, and induces caspase activation and leukemia cell death in the specific context of MCL-1 dependence. PMID:22999885

GS-nitroxide (JP4-039)-mediated radioprotection of human Fanconi anemia cell lines.

PubMed

Bernard, Mark E; Kim, Hyun; Berhane, Hebist; Epperly, Michael W; Franicola, Darcy; Zhang, Xichen; Houghton, Frank; Shields, Donna; Wang, Hong; Bakkenist, Christopher J; Frantz, Marie-Celine; Forbeck, Erin M; Goff, Julie P; Wipf, Peter; Greenberger, Joel S

2011-11-01

Fanconi anemia (FA) is an inherited disorder characterized by defective DNA repair and cellular sensitivity to DNA crosslinking agents. Clinically, FA is associated with high risk for marrow failure, leukemia and head and neck squamous cell carcinoma (HNSCC). Radiosensitivity in FA patients compromises the use of total-body irradiation for hematopoietic stem cell transplantation and radiation therapy for HNSCC. A radioprotector for the surrounding tissue would therefore be very valuable during radiotherapy for HNSCC. Clonogenic radiation survival curves were determined for pre- or postirradiation treatment with the parent nitroxide Tempol or JP4-039 in cells of four FA patient-derived cell lines and two transgene-corrected subclonal lines. FancG(-/-) (PD326) and FancD2(-/-) (PD20F) patient lines were more sensitive to the DNA crosslinking agent mitomycin C (MMC) than their transgene-restored subclonal cell lines (both P < 0.0001). FancD2(-/-) cells were more radiosensitive than the transgene restored subclonal cell line (ñ = 2.0 ± 0.7 and 4.7 ± 2.2, respectively, P = 0.03). In contrast, FancG(-/-) cells were radioresistant relative to the transgene-restored subclonal cell line (ñ = 9.4 ± 1.5 and 2.2 ± 05, respectively, P = 0.001). DNA strand breaks measured by the comet assay correlated with radiosensitivity. Cell lines from a Fanc-C and Fanc-A patients showed radiosensitivity similar to that of Fanc-D2(-/-) cells. A fluorophore-tagged JP4-039 (BODIPY-FL) analog targeted the mitochondria of the cell lines. Preirradiation or postirradiation treatment with JP4-039 at a lower concentration than Tempol significantly increased the radioresistance and stabilized the antioxidant stores of all cell lines. Tempol increased the toxicity of MMC in FancD2(-/-) cells. These data provide support for the potential clinical use of JP4-039 for normal tissue radioprotection during chemoradiotherapy in FA patients.

A novel Trojan-horse targeting strategy to reduce the non-specific uptake of nanocarriers by non-cancerous cells.

PubMed

Shen, Zheyu; Wu, Hao; Yang, Sugeun; Ma, Xuehua; Li, Zihou; Tan, Mingqian; Wu, Aiguo

2015-11-01

One big challenge with active targeting of nanocarriers is non-specific binding between targeting molecules and non-target moieties expressed on non-cancerous cells, which leads to non-specific uptake of nanocarriers by non-cancerous cells. Here, we propose a novel Trojan-horse targeting strategy to hide or expose the targeting molecules of nanocarriers on-demand. The non-specific uptake by non-cancerous cells can be reduced because the targeting molecules are hidden in hydrophilic polymers. The nanocarriers are still actively targetable to cancer cells because the targeting molecules can be exposed on-demand at tumor regions. Typically, Fe3O4 nanocrystals (FN) as magnetic resonance imaging (MRI) contrast agents were encapsulated into albumin nanoparticles (AN), and then folic acid (FA) and pH-sensitive polymers (PP) were grafted onto the surface of AN-FN to construct PP-FA-AN-FN nanoparticles. Fourier transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS), transmission electron microscope (TEM) and gel permeation chromatography (GPC) results confirm successful construction of PP-FA-AN-FN. According to difference of nanoparticle-cellular uptake between pH 7.4 and 5.5, the weight ratio of conjugated PP to nanoparticle FA-AN-FN (i.e. graft density) and the molecular weight of PP (i.e. graft length) are optimized to be 1.32 and 5.7 kDa, respectively. In vitro studies confirm that the PP can hide ligand FA to prevent it from binding to cells with FRα at pH 7.4 and shrink to expose FA at pH 5.5. In vivo studies demonstrate that our Trojan-horse targeting strategy can reduce the non-specific uptake of the PP-FA-AN-FN by non-cancerous cells. Therefore, our PP-FA-AN-FN might be used as an accurately targeted MRI contrast agent. Copyright © 2015 Elsevier Ltd. All rights reserved.

The impact of FANCD2 deficiency on formaldehyde-induced toxicity in human lymphoblastoid cell lines

PubMed Central

Ren, Xuefeng; Ji, Zhiying; McHale, Cliona M.; Yuh, Jessica; Bersonda, Jessica; Tang, Maycky; Smith, Martyn T.; Zhang, Luoping

2015-01-01

Formaldehyde (FA), a major industrial chemical and ubiquitous environmental pollutant, has recently been classified by the International Agency for Research on Cancer as a human leukemogen. The major mode of action of FA is thought to be the formation of DNA-protein crosslinks (DPCs). Repair of DPCs may be mediated by the Fanconi anemia pathway; however, data supporting the involvement of this pathway is limited, particularly in human hematopoietic cells. Therefore, we assessed the role of FANCD2, a critical component of the Fanconi anemia pathway, in FA-induced toxicity in human lymphoblast cell models of FANCD2-deficiency (PD20 cells) and FANCD2-sufficiency (PD20-D2 cells). After treatment of the cells with 0-150 μM FA for 24 hours, DPCs were increased in a dose-dependent manner in both cell lines, with greater increases in FANCD2-deficient PD20 cells. FA also induced cytotoxicity, micronuclei, chromosome aberrations, and apoptosis in a dose-dependent manner in both cell lines, with greater increases in cytotoxicity and apoptosis in PD20 cells. Increased levels of γ-ATR and γ-H2AX in both cell lines suggested the recognition of FA-induced DNA damage; however, the induction of BRCA2 was compromised in FANCD2-deficient PD20 cells, potentially reducing the capacity to repair DPCs. Together, these findings suggest that FANCD2 protein and the Fanconi anemia pathway are essential to protect human lymphoblastoid cells against FA toxicity. Future studies are needed to delineate the role of this pathway in mitigating FA-induced toxicity, particularly in hematopoietic stem cells, the target cells in leukemia. PMID:22872141

Lentiviral-mediated genetic correction of hematopoietic and mesenchymal progenitor cells from Fanconi anemia patients.

PubMed

Jacome, Ariana; Navarro, Susana; Río, Paula; Yañez, Rosa M; González-Murillo, Africa; Lozano, M Luz; Lamana, Maria Luisa; Sevilla, Julian; Olive, Teresa; Diaz-Heredia, Cristina; Badell, Isabel; Estella, Jesus; Madero, Luis; Guenechea, Guillermo; Casado, José; Segovia, Jose C; Bueren, Juan A

2009-06-01

Previous clinical trials based on the genetic correction of purified CD34(+) cells with gamma-retroviral vectors have demonstrated clinical efficacy in different monogenic diseases, including X-linked severe combined immunodeficiency, adenosine deaminase deficient severe combined immunodeficiency and chronic granulomatous disease. Similar protocols, however, failed to engraft Fanconi anemia (FA) patients with genetically corrected cells. In this study, we first aimed to correlate the hematological status of 27 FA patients with CD34(+) cell values determined in their bone marrow (BM). Strikingly, no correlation between these parameters was observed, although good correlations were obtained when numbers of colony-forming cells (CFCs) were considered. Based on these results, and because purified FA CD34(+) cells might have suboptimal repopulating properties, we investigated the possibility of genetically correcting unselected BM samples from FA patients. Our data show that the lentiviral transduction of unselected FA BM cells mediates an efficient phenotypic correction of hematopoietic progenitor cells and also of CD34(-) mesenchymal stromal cells (MSCs), with a reported role in hematopoietic engraftment. Our results suggest that gene therapy protocols appropriate for the treatment of different monogenic diseases may not be adequate for stem cell diseases like FA. We propose a new approach for the gene therapy of FA based on the rapid transduction of unselected hematopoietic grafts with lentiviral vectors (LVs).

Lentiviral-mediated Genetic Correction of Hematopoietic and Mesenchymal Progenitor Cells From Fanconi Anemia Patients

PubMed Central

Jacome, Ariana; Navarro, Susana; Río, Paula; Yañez, Rosa M; González-Murillo, Africa; Luz Lozano, M; Lamana, Maria Luisa; Sevilla, Julian; Olive, Teresa; Diaz-Heredia, Cristina; Badell, Isabel; Estella, Jesus; Madero, Luis; Guenechea, Guillermo; Casado, José; Segovia, Jose C; Bueren, Juan A

2009-01-01

Previous clinical trials based on the genetic correction of purified CD34+ cells with γ-retroviral vectors have demonstrated clinical efficacy in different monogenic diseases, including X-linked severe combined immunodeficiency, adenosine deaminase deficient severe combined immunodeficiency and chronic granulomatous disease. Similar protocols, however, failed to engraft Fanconi anemia (FA) patients with genetically corrected cells. In this study, we first aimed to correlate the hematological status of 27 FA patients with CD34+ cell values determined in their bone marrow (BM). Strikingly, no correlation between these parameters was observed, although good correlations were obtained when numbers of colony-forming cells (CFCs) were considered. Based on these results, and because purified FA CD34+ cells might have suboptimal repopulating properties, we investigated the possibility of genetically correcting unselected BM samples from FA patients. Our data show that the lentiviral transduction of unselected FA BM cells mediates an efficient phenotypic correction of hematopoietic progenitor cells and also of CD34− mesenchymal stromal cells (MSCs), with a reported role in hematopoietic engraftment. Our results suggest that gene therapy protocols appropriate for the treatment of different monogenic diseases may not be adequate for stem cell diseases like FA. We propose a new approach for the gene therapy of FA based on the rapid transduction of unselected hematopoietic grafts with lentiviral vectors (LVs). PMID:19277017

Rac1 Recruitment to the Archipelago Structure of the Focal Adhesion through the Fluid Membrane as Revealed by Single-Molecule Analysis

PubMed Central

Shibata, Akihiro C E; Chen, Limin H; Nagai, Rie; Ishidate, Fumiyoshi; Chadda, Rahul; Miwa, Yoshihiro; Naruse, Keiji; Shirai, Yuki M; Fujiwara, Takahiro K; Kusumi, Akihiro

2013-01-01

The focal adhesion (FA) is an integrin-based structure built in/on the plasma membrane (PM), linking the extracellular matrix to the actin stress-fibers, working as cell migration scaffolds. Previously, we proposed the archipelago architecture of the FA, in which FA largely consists of fluid membrane, dotted with small islands accumulating FA proteins: membrane molecules enter the inter-island channels in the FA zone rather freely, and the integrins in the FA-protein islands rapidly exchanges with those in the bulk membrane. Here, we examined how Rac1, a small G-protein regulating FA formation, and its activators αPIX and βPIX, are recruited to the FA zones. PIX molecules are recruited from the cytoplasm to the FA zones directly. In contrast, majorities of Rac1 molecules first arrive from the cytoplasm on the general inner PM surface, and then enter the FA zones via lateral diffusion on the PM, which is possible due to rapid Rac1 diffusion even within the FA zones, slowed only by a factor of two to four compared with that outside. The constitutively-active Rac1 mutant exhibited temporary and all-time immobilizations in the FA zone, suggesting that upon PIX-induced Rac1 activation at the FA-protein islands, Rac1 tends to be immobilized at the FA-protein islands. © 2013 Wiley Periodicals, Inc PMID:23341328

76 FR 73760 - In the Matter of the Designation of Imad Fa'iz Mughniyah also Known as Imad Fayiz Mughniyah as a...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-29

... DEPARTMENT OF STATE [Public Notice 7699] In the Matter of the Designation of Imad Fa'iz Mughniyah also Known as Imad Fayiz Mughniyah as a Specially Designated Global Terrorist Pursuant to Section 1(b... amended (``the Order''), I hereby determine that the individual known as Imad Fa'iz Mughniyah, also known...

Targeted gene therapy and cell reprogramming in Fanconi anemia

PubMed Central

Rio, Paula; Baños, Rocio; Lombardo, Angelo; Quintana-Bustamante, Oscar; Alvarez, Lara; Garate, Zita; Genovese, Pietro; Almarza, Elena; Valeri, Antonio; Díez, Begoña; Navarro, Susana; Torres, Yaima; Trujillo, Juan P; Murillas, Rodolfo; Segovia, Jose C; Samper, Enrique; Surralles, Jordi; Gregory, Philip D; Holmes, Michael C; Naldini, Luigi; Bueren, Juan A

2014-01-01

Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies. PMID:24859981

Use of OCTA, FA, and Ultra-Widefield Imaging in Quantifying Retinal Ischemia: A Review.

PubMed

Or, Chris; Sabrosa, Almyr S; Sorour, Osama; Arya, Malvika; Waheed, Nadia

2018-01-01

As ischemia remains a key prognostic factor in the management of various diseases including diabetic retinopathy, an increasing amount of research has been dedicated to its quantification as a potential biomarker. Advancements in the quantification of retinal ischemia have been made with the imaging modalities of fluorescein angiography (FA), ultra-widefield imaging (UWF), and optical coherence tomography angiography (OCTA), with each imaging modality offering certain benefits over the others. FA remains the gold standard in assessing the extent of ischemia. UWF imaging has allowed for the assessment of peripheral ischemia via FA. It is, however, OCTA that offers the best visualization of retinal vasculature with its noninvasive depth-resolved imaging and therefore has the potential to become a mainstay in the assessment of retinal ischemia. The primary purpose of this article is to review the use of FA, UWF, and OCTA to quantify retinal ischemia and the various methods described in the literature by which this is achieved. Copyright 2018 Asia-Pacific Academy of Ophthalmology.

Autophagy regulates cisplatin-induced stemness and chemoresistance via the upregulation of CD44, ABCB1 and ADAM17 in oral squamous cell carcinoma.

PubMed

Naik, Prajna Paramita; Mukhopadhyay, Subhadip; Panda, Prashanta Kumar; Sinha, Niharika; Das, Chandan Kanta; Mishra, Rajakishore; Patil, Shankargouda; Bhutia, Sujit Kumar

2018-02-01

We inspected the relevance of CD44, ABCB1 and ADAM17 in OSCC stemness and deciphered the role of autophagy/mitophagy in regulating stemness and chemoresistance. A retrospective analysis of CD44, ABCB1 and ADAM17 with respect to the various clinico-pathological factors and their correlation was analysed in sixty OSCC samples. Furthermore, the stemness and chemoresistance were studied in resistant oral cancer cells using sphere formation assay, flow cytometry and florescence microscopy. The role of autophagy/mitophagy was investigated by transient transfection of siATG14, GFP-LC3, tF-LC3, mKeima-Red-Mito7 and Western blot analysis of autophagic and mitochondrial proteins. In OSCC, high CD44, ABCB1 and ADAM17 expressions were correlated with higher tumour grades and poor differentiation and show significant correlation in their co-expression. In vitro and OSCC tissue double labelling confirmed that CD44 + cells co-expresses ABCB1 and ADAM17. Further, cisplatin (CDDP)-resistant FaDu cells displayed stem-like features and higher CD44, ABCB1 and ADAM17 expression. Higher autophagic flux and mitophagy were observed in resistant FaDu cells as compared to parental cells, and inhibition of autophagy led to the decrease in stemness, restoration of mitochondrial proteins and reduced expression of CD44, ABCB1 and ADAM17. The CD44 + /ABCB1 + /ADAM17 + expression in OSCC is associated with stemness and chemoresistance. Further, this study highlights the involvement of mitophagy in chemoresistance and autophagic regulation of stemness in OSCC. © 2017 John Wiley & Sons Ltd.

The IGF-1 Receptor Identifies a Pool of Human Cardiac Stem Cells with Superior Therapeutic Potential for Myocardial Regeneration

PubMed Central

D’Amario, Domenico; Cabral-Da-Silva, Mauricio; Zheng, Hanqiao; Fiorini, Claudia; Goichberg, Polina; Steadman, Elisabeth; Ferreira-Martins, João; Sanada, Fumihiro; Piccoli, Marco; Cappetta, Donato; D’Alessandro, David A.; Michler, Robert E.; Hosoda, Toru; Anastasia, Luigi; Rota, Marcello; Leri, Annarosa; Anversa, Piero; Kajstura, Jan

2012-01-01

Rationale Age and coronary artery disease may negatively affect the function of human cardiac stem cells (hCSCs) and their potential therapeutic efficacy for autologous cell transplantation in the failing heart. Objective Insulin-like growth factor 1 (IGF-1) and 2 (IGF-2), and angiotensin II (Ang II) and their receptors, IGF-1R, IGF-2R and AT1R, were characterized in c-kit-positive-hCSCs to establish whether these systems would allow us to separate hCSC classes with different growth reserve in the aging and diseased myocardium. Methods and Results C-kit-positive-hCSCs were collected from myocardial samples obtained from 24 patients, 48 to 86 years of age, undergoing elective cardiac surgery for coronary artery disease. The expression of IGF-1R in hCSCs recognized a young cell phenotype defined by long telomeres, high telomerase activity, enhanced cell proliferation and attenuated apoptosis. In addition to IGF-1, IGF-1R-positive-hCSCs secreted IGF-2 that promoted myocyte differentiation. Conversely, the presence of IGF-2R and AT1R, in the absence of IGF-1R, identified senescent hCSCs with impaired growth reserve and increased susceptibility to apoptosis. The ability of IGF-1R-positive-hCSCs to regenerate infarcted myocardium was then compared with that of unselected c-kit-positive-hCSCs. IGF-1R-positive-hCSCs improved cardiomyogenesis and vasculogenesis. Pretreatment of IGF-1R-positive-hCSCs with IGF-2 resulted in the formation of more mature myocytes and superior recovery of ventricular structure. Conclusions hCSCs expressing only IGF-1R synthesize both IGF-1 and IGF-2, which are potent modulators of stem cell replication, commitment to the myocyte lineage and myocyte differentiation, pointing to this hCSC subset as the ideal candidate cell for the management of human heart failure. PMID:21546606

Aeroelastic modeling for the FIT team F/A-18 simulation

NASA Technical Reports Server (NTRS)

Zeiler, Thomas A.; Wieseman, Carol D.

1989-01-01

Some details of the aeroelastic modeling of the F/A-18 aircraft done for the Functional Integration Technology (FIT) team's research in integrated dynamics modeling and how these are combined with the FIT team's integrated dynamics model are described. Also described are mean axis corrections to elastic modes, the addition of nonlinear inertial coupling terms into the equations of motion, and the calculation of internal loads time histories using the integrated dynamics model in a batch simulation program. A video tape made of a loads time history animation was included as a part of the oral presentation. Also discussed is work done in one of the areas of unsteady aerodynamic modeling identified as needing improvement, specifically, in correction factor methodologies for improving the accuracy of stability derivatives calculated with a doublet lattice code.

Adaptive Augmenting Control Flight Characterization Experiment on an F/A-18

NASA Technical Reports Server (NTRS)

VanZwieten, Tannen S.; Orr, Jeb S.; Wall, John H.; Gilligan, Eric T.

2014-01-01

(see Figure 1). The MSFC algorithm design was formulated during the Constellation Program and reached a high maturity level during SLS through simulation-based development and internal and external analytical review. The AAC algorithm design has three summary-level objectives: (1) "Do no harm;" return to baseline control design when not needed, (2) Increase performance; respond to error in ability of vehicle to track command, and (3) Regain stability; respond to undesirable control-structure interaction or other parasitic dynamics. AAC has been successfully implemented as part of the Space Launch System baseline design, including extensive testing in high-fidelity 6-DOF simulations the details of which are described in [1]. The Dryden Flight Research Center's F/A-18 Full-Scale Advanced Systems Testbed (FAST) platform is used to conduct an algorithm flight characterization experiment intended to fully vet the aforementioned design objectives. FAST was specifically designed with this type of test program in mind. The onboard flight control system has full-authority experiment control of ten aerodynamic effectors and two throttles. It has production and research sensor inputs and pilot engage/disengage and real-time configuration of up to eight different experiments on a single flight. It has failure detection and automatic reversion to fail-safe mode. The F/A-18 aircraft has an experiment envelope cleared for full-authority control and maneuvering and exhibits characteristics for robust recovery from unusual attitudes and configurations aided by the presence of a qualified test pilot. The F/A-18 aircraft has relatively high mass and inertia with exceptional performance; the F/A-18 also has a large thrust-to-weight ratio, owing to its military heritage. This enables the simulation of a portion of the ascent trajectory with a high degree of dynamic similarity to a launch vehicle, and the research flight control system can simulate unstable longitudinal dynamics. Parasitic

Deficiency of UBE2T, the E2 Ubiquitin Ligase Necessary for FANCD2 and FANCI Ubiquitination, Causes FA-T Subtype of Fanconi Anemia.

PubMed

Rickman, Kimberly A; Lach, Francis P; Abhyankar, Avinash; Donovan, Frank X; Sanborn, Erica M; Kennedy, Jennifer A; Sougnez, Carrie; Gabriel, Stacey B; Elemento, Olivier; Chandrasekharappa, Settara C; Schindler, Detlev; Auerbach, Arleen D; Smogorzewska, Agata

2015-07-07

Fanconi anemia (FA) is a rare bone marrow failure and cancer predisposition syndrome resulting from pathogenic mutations in genes encoding proteins participating in the repair of DNA interstrand crosslinks (ICLs). Mutations in 17 genes (FANCA-FANCS) have been identified in FA patients, defining 17 complementation groups. Here, we describe an individual presenting with typical FA features who is deficient for the ubiquitin-conjugating enzyme (E2), UBE2T. UBE2T is known to interact with FANCL, the E3 ubiquitin-ligase component of the multiprotein FA core complex, and is necessary for the monoubiquitination of FANCD2 and FANCI. Proband fibroblasts do not display FANCD2 and FANCI monoubiquitination, do not form FANCD2 foci following treatment with mitomycin C, and are hypersensitive to crosslinking agents. These cellular defects are complemented by expression of wild-type UBE2T, demonstrating that deficiency of the protein UBE2T can lead to Fanconi anemia. UBE2T gene gains an alias of FANCT. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Fatty acid-binding protein 5 (FABP5) promotes lipolysis of lipid droplets, de novo fatty acid (FA) synthesis and activation of nuclear factor-kappa B (NF-κB) signaling in cancer cells.

PubMed

Senga, Shogo; Kobayashi, Narumi; Kawaguchi, Koichiro; Ando, Akira; Fujii, Hiroshi

2018-06-12

Fatty acid-binding proteins (FABPs) are involved in binding and storing hydrophobic ligands such as long-chain fatty acids, as well as transporting them to the appropriate compartments in the cell. Epidermal fatty acid-binding protein (FABP5) is an intracellular lipid-binding protein that is abundantly expressed in adipocytes and macrophages. Previous studies have revealed that the FABP5 expression level is closely related to malignancy in various types of cancer. However, its precise functions in the metabolisms of cancer cells remain unclear. Here, we revealed that FABP5 knockdown significantly induced downregulation of the genes expression, such as hormone-sensitive lipase (HSL), monoacylglycerol lipase (MAGL), elongation of long-chain fatty acid member 6 (Elovl6), and acyl-CoA synthetase long-chain family member 1 (ACSL1), which are involved in altered lipid metabolism, lipolysis, and de novo FA synthesis in highly aggressive prostate and breast cancer cells. Moreover, we demonstrated that FABP5 induced inflammation and cytokine production through the nuclear factor-kappa B signaling pathway activated by reactive oxygen species and protein kinase C in PC-3 and MDA-MB-231 cells. Thus, FABP5 might regulate lipid quality and/or quantity to promote aggressiveness such as cell growth, invasiveness, survival, and inflammation in prostate and breast cancer cells. In the present study, we have revealed for the first time that high expression of FABP5 plays a critical role in alterations of lipid metabolism, leading to cancer development and metastasis in highly aggressive prostate and breast cancer cells. Copyright © 2018. Published by Elsevier B.V.

In cell mutational interference mapping experiment (in cell MIME) identifies the 5' polyadenylation signal as a dual regulator of HIV-1 genomic RNA production and packaging.

PubMed

Smyth, Redmond P; Smith, Maureen R; Jousset, Anne-Caroline; Despons, Laurence; Laumond, Géraldine; Decoville, Thomas; Cattenoz, Pierre; Moog, Christiane; Jossinet, Fabrice; Mougel, Marylène; Paillart, Jean-Christophe; von Kleist, Max; Marquet, Roland

2018-05-18

Non-coding RNA regulatory elements are important for viral replication, making them promising targets for therapeutic intervention. However, regulatory RNA is challenging to detect and characterise using classical structure-function assays. Here, we present in cell Mutational Interference Mapping Experiment (in cell MIME) as a way to define RNA regulatory landscapes at single nucleotide resolution under native conditions. In cell MIME is based on (i) random mutation of an RNA target, (ii) expression of mutated RNA in cells, (iii) physical separation of RNA into functional and non-functional populations, and (iv) high-throughput sequencing to identify mutations affecting function. We used in cell MIME to define RNA elements within the 5' region of the HIV-1 genomic RNA (gRNA) that are important for viral replication in cells. We identified three distinct RNA motifs controlling intracellular gRNA production, and two distinct motifs required for gRNA packaging into virions. Our analysis reveals the 73AAUAAA78 polyadenylation motif within the 5' PolyA domain as a dual regulator of gRNA production and gRNA packaging, and demonstrates that a functional polyadenylation signal is required for viral packaging even though it negatively affects gRNA production.

s-SHIP expression identifies a subset of murine basal prostate cells as neonatal stem cells

PubMed Central

Brocqueville, Guillaume; Chmelar, Renee S.; Bauderlique-Le Roy, Hélène; Deruy, Emeric; Tian, Lu; Vessella, Robert L.; Greenberg, Norman M.; Bourette, Roland P.

2016-01-01

Isolation of prostate stem cells (PSCs) is crucial for understanding their biology during normal development and tumorigenesis. In this aim, we used a transgenic mouse model expressing GFP from the stem cell-specific s-SHIP promoter to mark putative stem cells during postnatal prostate development. Here we show that cells identified by GFP expression are present transiently during early prostate development and localize to the basal cell layer of the epithelium. These prostate GFP+ cells are a subpopulation of the Lin− CD24+ Sca-1+ CD49f+ cells and are capable of self-renewal together with enhanced growth potential in sphere-forming assay in vitro, a phenotype consistent with that of a PSC population. Transplantation assays of prostate GFP+ cells demonstrate reconstitution of prostate ducts containing both basal and luminal cells in renal grafts. Altogether, these results demonstrate that s-SHIP promoter expression is a new marker for neonatal basal prostate cells exhibiting stem cell properties that enables PSCs in situ identification and isolation via a single consistent parameter. Transcriptional profiling of these GFP+ neonatal stem cells showed an increased expression of several components of the Wnt signaling pathway. It also identified stem cell regulators with potential applications for further analyses of normal and cancer stem cells. PMID:27081082

Superparamagnetic poly(methyl methacrylate) nanoparticles surface modified with folic acid presenting cell uptake mediated by endocytosis

NASA Astrophysics Data System (ADS)

Feuser, Paulo Emilio; Jacques, Amanda Virtuoso; Arévalo, Juan Marcelo Carpio; Rocha, Maria Eliane Merlin; dos Santos-Silva, Maria Claudia; Sayer, Claudia; de Araújo, Pedro H. Hermes

2016-04-01

The encapsulation of superparamagnetic nanoparticles (MNPs) in polymeric nanoparticles (NPs) with modified surfaces can improve targeted delivery and induce cell death by hyperthermia. The goals of this study were to synthesize and characterize surface modified superparamagnetic poly(methyl methacrylate) with folic acid (FA) prepared by miniemulsion polymerization (MNPsPMMA-FA) and to evaluate their in vitro cytotoxicity and cellular uptake in non-tumor cells, murine fibroblast (L929) cells and tumor cells that overexpressed folate receptor (FR) β, and chronic myeloid leukemia cells in blast crisis (K562). Lastly, hemolysis assays were performed on human red blood cells. MNPsPMMA-FA presented an average mean diameter of 135 nm and a saturation magnetization (Ms) value of 37 emu/g of iron oxide, as well as superparamagnetic behavior. The MNPsPMMA-FA did not present cytotoxicity in L929 and K562 cells. Cellular uptake assays showed a higher uptake of MNPsPMMA-FA than MNPsPMMA in K562 cells when incubated at 37 °C. On the other hand, MNPsPMMA-FA showed a low uptake when endocytosis mechanisms were blocked at low temperature (4 °C), suggesting that the MNPsPMMA-FA uptake was mediated by endocytosis. High concentrations of MNPsPMMA-FA showed hemocompatibility when incubated for 24 h in human red blood cells. Therefore, our results suggest that these carrier systems can be an excellent alternative in targeted drug delivery via FR.

Stability analysis of an F/A-18 E/F cable mount m odel

NASA Technical Reports Server (NTRS)

Thompson, Nancy; Farmer, Moses

1994-01-01

A full-span F/A-18 E/F cable mounted wind tunnel model is part of a flutter clearance program at the NASA Langley Transonic Dynamics Tunnel. Parametric analysis of this model using GRUMCBL software was conducted to assess stability for wind tunnel tests. Two configurations of the F/A-18 E/F were examined. The parameters examined were pulley-cable friction, mach number, dynamic pressure, cable geometry, center of gravity location, cable tension, snubbing the model, drag, and test medium. For the nominal cable geometry (Cable Geometry 1), Configuration One was unstable for cases with higher pulley-cable friction coefficients. A new cable geometry (Cable Geometry 3) was determined in which Configuration One was stable for all cases evaluated. Configuration Two with the nominal center of gravity position was found to be unstable for cases with higher pulley-cable friction coefficients; however, the model was stable when the center of gravity moved forward 1/2. The model was tested using the cable mount system during the initial wind tunnel entry and was stable as predicted.

Measuring X-ray anisotropy in solar flares. Prospective stereoscopic capabilities of STIX and MiSolFA

NASA Astrophysics Data System (ADS)

Casadei, Diego; Jeffrey, Natasha L. S.; Kontar, Eduard P.

2017-09-01

Context. During a solar flare, a large percentage of the magnetic energy released goes into the kinetic energy of non-thermal particles, with X-ray observations providing a direct connection to keV flare-accelerated electrons. However, the electron angular distribution, a prime diagnostic tool of the acceleration mechanism and transport, is poorly known. Aims: During the next solar maximum, two upcoming space-borne X-ray missions, STIX on board Solar Orbiter and MiSolFA, will perform stereoscopic X-ray observations of solar flares at two different locations: STIX at 0.28 AU (at perihelion) and up to inclinations of 25°, and MiSolFA in a low-Earth orbit. The combined observations from these cross-calibrated detectors will allow us to infer the electron anisotropy of individual flares confidently for the first time. Methods: We simulated both instrumental and physical effects for STIX and MiSolFA including thermal shielding, background and X-ray Compton backscattering (albedo effect) in the solar photosphere. We predict the expected number of observable flares available for stereoscopic measurements during the next solar maximum. We also discuss the range of useful spacecraft observation angles for the challenging case of close-to-isotropic flare anisotropy. Results: The simulated results show that STIX and MiSolFA will be capable of detecting low levels of flare anisotropy, for M1-class or stronger flares, even with a relatively small spacecraft angular separation of 20-30°. Both instruments will directly measure the flare X-ray anisotropy of about 40 M- and X-class solar flares during the next solar maximum. Conclusions: Near-future stereoscopic observations with Solar Orbiter/STIX and MiSolFA will help distinguishing between competing flare-acceleration mechanisms, and provide essential constraints regarding collisional and non-collisional transport processes occurring in the flaring atmosphere for individual solar flares.

System analysis identifies distinct and common functional networks governed by transcription factor ASCL1, in glioma and small cell lung cancer.

PubMed

Donakonda, Sainitin; Sinha, Swati; Dighe, Shrinivas Nivrutti; Rao, Manchanahalli R Satyanarayana

2017-07-25

ASCL1 is a basic Helix-Loop-Helix transcription factor (TF), which is involved in various cellular processes like neuronal development and signaling pathways. Transcriptome profiling has shown that ASCL1 overexpression plays an important role in the development of glioma and Small Cell Lung Carcinoma (SCLC), but distinct and common molecular mechanisms regulated by ASCL1 in these cancers are unknown. In order to understand how it drives the cellular functional network in these two tumors, we generated a gene expression profile in a glioma cell line (U87MG) to identify ASCL1 gene targets by an si RNA silencing approach and then compared this with a publicly available dataset of similarly silenced SCLC (NCI-H1618 cells). We constructed TF-TF and gene-gene interactions, as well as protein interaction networks of ASCL1 regulated genes in glioma and SCLC cells. Detailed network analysis uncovered various biological processes governed by ASCL1 target genes in these two tumor cell lines. We find that novel ASCL1 functions related to mitosis and signaling pathways influencing development and tumor growth are affected in both glioma and SCLC cells. In addition, we also observed ASCL1 governed functional networks that are distinct to glioma and SCLC.

UV-triggered Affinity Capture Identifies Interactions between the Plasmodium falciparum Multidrug Resistance Protein 1 (PfMDR1) and Antimalarial Agents in Live Parasitized Cells*

PubMed Central

Brunner, Ralf; Ng, Caroline L.; Aissaoui, Hamed; Akabas, Myles H.; Boss, Christoph; Brun, Reto; Callaghan, Paul S.; Corminboeuf, Olivier; Fidock, David A.; Frame, Ithiel J.; Heidmann, Bibia; Le Bihan, Amélie; Jenö, Paul; Mattheis, Corinna; Moes, Suzette; Müller, Ingrid B.; Paguio, Michelle; Roepe, Paul D.; Siegrist, Romain; Voss, Till; Welford, Richard W. D.; Wittlin, Sergio; Binkert, Christoph

2013-01-01

A representative of a new class of potent antimalarials with an unknown mode of action was recently described. To identify the molecular target of this class of antimalarials, we employed a photo-reactive affinity capture method to find parasite proteins specifically interacting with the capture compound in living parasitized cells. The capture reagent retained the antimalarial properties of the parent molecule (ACT-213615) and accumulated within parasites. We identified several proteins interacting with the capture compound and established a functional interaction between ACT-213615 and PfMDR1. We surmise that PfMDR1 may play a role in the antimalarial activity of the piperazine-containing compound ACT-213615. PMID:23754276

Slicing Method for curved façade and window extraction from point clouds

NASA Astrophysics Data System (ADS)

Iman Zolanvari, S. M.; Laefer, Debra F.

2016-09-01

Laser scanning technology is a fast and reliable method to survey structures. However, the automatic conversion of such data into solid models for computation remains a major challenge, especially where non-rectilinear features are present. Since, openings and the overall dimensions of the buildings are the most critical elements in computational models for structural analysis, this article introduces the Slicing Method as a new, computationally-efficient method for extracting overall façade and window boundary points for reconstructing a façade into a geometry compatible for computational modelling. After finding a principal plane, the technique slices a façade into limited portions, with each slice representing a unique, imaginary section passing through a building. This is done along a façade's principal axes to segregate window and door openings from structural portions of the load-bearing masonry walls. The method detects each opening area's boundaries, as well as the overall boundary of the façade, in part, by using a one-dimensional projection to accelerate processing. Slices were optimised as 14.3 slices per vertical metre of building and 25 slices per horizontal metre of building, irrespective of building configuration or complexity. The proposed procedure was validated by its application to three highly decorative, historic brick buildings. Accuracy in excess of 93% was achieved with no manual intervention on highly complex buildings and nearly 100% on simple ones. Furthermore, computational times were less than 3 sec for data sets up to 2.6 million points, while similar existing approaches required more than 16 hr for such datasets.

Combined cistrome and transcriptome analysis of SKI in AML cells identifies SKI as a co-repressor for RUNX1

PubMed Central

Feld, Christine; Sahu, Peeyush; Frech, Miriam; Finkernagel, Florian; Nist, Andrea; Stiewe, Thorsten; Bauer, Uta-Maria; Neubauer, Andreas

2018-01-01

Abstract SKI is a transcriptional co-regulator and overexpressed in various human tumors, for example in acute myeloid leukemia (AML). SKI contributes to the origin and maintenance of the leukemic phenotype. Here, we use ChIP-seq and RNA-seq analysis to identify the epigenetic alterations induced by SKI overexpression in AML cells. We show that approximately two thirds of differentially expressed genes are up-regulated upon SKI deletion, of which >40% harbor SKI binding sites in their proximity, primarily in enhancer regions. Gene ontology analysis reveals that many of the differentially expressed genes are annotated to hematopoietic cell differentiation and inflammatory response, corroborating our finding that SKI contributes to a myeloid differentiation block in HL60 cells. We find that SKI peaks are enriched for RUNX1 consensus motifs, particularly in up-regulated SKI targets upon SKI deletion. RUNX1 ChIP-seq displays that nearly 70% of RUNX1 binding sites overlap with SKI peaks, mainly at enhancer regions. SKI and RUNX1 occupy the same genomic sites and cooperate in gene silencing. Our work demonstrates for the first time the predominant co-repressive function of SKI in AML cells on a genome-wide scale and uncovers the transcription factor RUNX1 as an important mediator of SKI-dependent transcriptional repression. PMID:29471413

Folic acid-decorated polyamidoamine dendrimer mediates selective uptake and high expression of genes in head and neck cancer cells.

PubMed

Xu, Leyuan; Kittrell, Shannon; Yeudall, W Andrew; Yang, Hu

2016-11-01

Folic acid (FA)-decorated polyamidoamine dendrimer G4 (G4-FA) was synthesized and studied for targeted delivery of genes to head and neck cancer cells expressing high levels of folate receptors (FRs). Cellular uptake, targeting specificity, cytocompatibility and transfection efficiency were evaluated. G4-FA competes with free FA for the same binding site. G4-FA facilitates the cellular uptake of DNA plasmids in a FR-dependent manner and selectively delivers plasmids to FR-high cells, leading to enhanced gene expression. G4-FA is a suitable vector to deliver genes selectively to head and neck cancer cells. The fundamental understandings of G4-FA as a vector and its encouraging transfection results for head and neck cancer cells provided support for its further testing in vivo.

Characterization and anticancer potential of ferulic acid-loaded chitosan nanoparticles against ME-180 human cervical cancer cell lines

NASA Astrophysics Data System (ADS)

Panwar, Richa; Sharma, Asvene K.; Kaloti, Mandeep; Dutt, Dharm; Pruthi, Vikas

2016-08-01

Ferulic acid (FA) is a widely distributed hydroxycinnamic acid found in various cereals and fruits exhibiting potent antioxidant and anticancer activities. However, due to low solubility and permeability, its availability to biological systems is limited. Non-toxic chitosan-tripolyphosphate pentasodium (CS-TPP) nanoparticles (NPs) are used to load sparingly soluble molecules and drugs, increasing their bioavailability. In the present work, we have encapsulated FA into the CS-TPP NPs to increase its potential as a therapeutic agent. Different concentrations of FA were tested to obtain optimum sized FA-loaded CS-TPP nanoparticles (FA/CS-TPP NPs) by ionic gelation method. Nanoparticles were characterized by scanning electron microscopy, Fourier transformation infrared spectroscopy (FTIR), thermogravimetric analyses and evaluated for their anticancer activity against ME-180 human cervical cancer cell lines. The FTIR spectra confirmed the encapsulation of FA and thermal analysis depicted its degradation profile. A concentration-dependent relationship between FA encapsulation efficiency and FA/CS-TPP NPs diameter was observed. Smooth and spherical FA-loaded cytocompatible nanoparticles with an average diameter of 125 nm were obtained at 40 µM FA conc. The cytotoxicity of 40 µM FA/CS-TPP NPs against ME-180 cervical cancer cell lines was found to be higher as compared to 40 µM native FA. Apoptotic morphological changes as cytoplasmic remnants and damaged wrinkled cells in ME-180 cells were visualized using scanning electron microscopic and fluorescent microscopic techniques. Data concluded that chitosan enveloped FA nanoparticles could be exploited as an excellent therapeutic drug against cancer cells proliferation.

Disrupting Façades of Clarity in the Teaching and Learning of Qualitative Research

ERIC Educational Resources Information Center

Carducci, Rozana; Pasque, Penny A.; Kuntz, Aaron M.; Contreras-­McGavin, Melissa

2013-01-01

In this article we examine two methodological façades of clarity that commonly shroud critical qualitative educational inquiry. More specifically, we interrogate discussions of reflexivity and positionality and explore the ways in which methodology curricula and instructional practices perpetuate façades of clarity, or a false sense of coherence,…

Mixed compared with single-source proteins in high-protein diets affect kidney structure and function differentially in obese fa/fa Zucker rats.

PubMed

Devassy, Jessay G; Wojcik, Jennifer L; Ibrahim, Naser H M; Zahradka, Peter; Taylor, Carla G; Aukema, Harold M

2017-02-01

Questions remain regarding the potential negative effects of dietary high protein (HP) on kidney health, particularly in the context of obesity in which the risk for renal disease is already increased. To examine whether some of the variability in HP effects on kidney health may be due to source of protein, obese fa/fa Zucker rats were given HP (35% of energy from protein) diets containing either casein, soy protein, or a mixed source of animal and plant proteins for 12 weeks. Control lean and obese rats were given diets containing casein at normal protein (15% of energy from protein) levels. Body weight and blood pressure were measured, and markers of renal structural changes, damage, and function were assessed. Obesity alone resulted in mild renal changes, as evidenced by higher kidney weights, proteinuria, and glomerular volumes. In obese rats, increasing the protein level using the single, but not mixed, protein sources resulted in higher renal fibrosis compared with the lean rats. The mixed-protein HP group also had lower levels of serum monocyte chemoattractant protein-1, even though this diet further increased kidney and glomerular size. Soy and mixed-protein HP diets also resulted in a small number of damaged glomeruli, while soy compared with mixed-protein HP diet delayed the increase in blood pressure over time. Since obesity itself confers added risk of renal disease, an HP diet from mixed-protein sources that enables weight loss but has fewer risks to renal health may be advantageous.

Stem Cell Genetic Therapy for Fanconi Anemia - A New Hope.

PubMed

Hanenberg, Helmut; Roellecke, Katharina; Wiek, Constanze

2017-01-01

Fanconi anemia (FA) is a rare inherited DNA disorder clinically characterized by congenital malformations, progressive bone marrow failure, and cancer susceptibility. Due to a strong survival advantage of spontaneously corrected 'normal' hematopoietic stem cells (HSCs) in a few patients, FA is considered a model disorder for genetic correction of autologous stem cells, where genetically corrected stem cells and their progeny have a strong in vivo selective advantage, ultimately leading to normal hematopoiesis. Despite these apparently ideal circumstances, three HSC gene therapy trials with gammaretroviral vectors (stage I) designed to cure the hematological manifestation of FA completely failed to provide long-term clinical benefits for patients, predominantly due to the combination of insufficient gene transfer technologies and incompletely understood FA HSC pathobiology. Currently, FA gene therapy is in stage II where, based on an improved understanding of the cellular defects in FA HSCs, consequently adapted transduction protocols are being used in two phase I/II trials for in vitro genetic correction of FANCA-deficient hematopoietic stem cells. These results are eagerly awaited. Independent from the outcome of these studies, technologies are already available that seem highly attractive for testing in FA. In stage III, this would ultimately include targeted in vivo correction of autologous HSCs by overexpression of nonintegrating lentiviral vectors with scaffold/matrix attachment region elements using specific envelopes as pseudotypes. Although currently still challenging, in a few years in vivo genome editing approaches will be readily available in stage IV, in which the delivery of the editing machinery/ complex is targeted to the autologous FA HSCs by the nonintegrating lentiviral vectors established in stage III. Even low levels of corrected stem cells will then quickly repopulate the entire hematopoiesis of the patient. We therefore are sanguine that in