scholarly journals Involvement of Arabidopsis Multi-Copper Oxidase-Encoding LACCASE12 in Root-to-Shoot Iron Partitioning: A Novel Example of Copper-Iron Crosstalk

2021 ◽  
Vol 12 ◽  
Author(s):  
María Bernal ◽  
Ute Krämer
Keyword(s):  
Current Knowledge ◽  
Essential Elements ◽  
Fe Deficiency ◽  
Phenotypic Characterization ◽  
Cu Deficiency ◽  
Stress Protection ◽  
Ferroxidase Activity ◽  
Fe Homeostasis

Numerous central biological processes depend on the participation of the essential elements iron (Fe) or copper (Cu), including photosynthesis, respiration, cell wall remodeling and oxidative stress protection. Yet, both Fe and Cu metal cations can become toxic when accumulated in excess. Because of the potent ligand-binding and redox chemistries of these metals, there is a need for the tight and combined homeostatic control of their uptake and distribution. Several known examples pinpoint an inter-dependence of Fe and Cu homeostasis in eukaryotes, mostly in green algae, yeast and mammals, but this is less well understood in multicellular plants to date. In Arabidopsis, Cu deficiency causes secondary Fe deficiency, and this is associated with reduced in vitro ferroxidase activity and decreased root-to-shoot Fe translocation. Here we summarize the current knowledge of the cross-talk between Cu and Fe homeostasis and present a partial characterization of LACCASE12 (LAC12) that encodes a member of the multicopper oxidase (MCO) protein family in Arabidopsis. LAC12 transcript levels increase under Fe deficiency. The phenotypic characterization of two mutants carrying T-DNA insertions suggests a role of LAC12 in root-to-shoot Fe partitioning and in maintaining growth on Fe-deficient substrates. A molecular understanding of the complex interactions between Fe and Cu will be important for combating Fe deficiency in crops and for advancing biofortification approaches.

scholarly journals Isolation, cultivation and immunofluorescence characterization of lamellar keratinocytes from equine hoof by using explants

2019 ◽  
Vol 39 (4) ◽  
pp. 292-298
Author(s):  
João P.H. Pfeifer ◽  
Vitor H. Santos ◽  
Gustavo Rosa ◽  
Jaqueline B. Souza ◽  
Marcos Jun Watanabe ◽  
...  
Keyword(s):  
Current Knowledge ◽  
Primary Cultures ◽  
Culture Method ◽  
Ki 67 ◽  
Keratinocyte Culture ◽  
Cell Growth And Differentiation ◽  
Equine Hoof ◽  
Horse Health

ABSTRACT: The importance of the hoof to the horse health is clear, and the current knowledge regarding the cellular aspects of hoof keratinocytes is poor. Studies on equine keratinocyte culture are scarce. Developing keratinocyte cultures in vitro is a condition for studies on molecular biology, cell growth and differentiation. Some methods have already been established, such as those for skin keratinocyte culture. However, few methodologies are found for lamellar keratinocytes. The objective of this study was to standardize the equine hoof keratinocyte isolation and cultivation, and then characterize the cell immunophenotype. For this, the primary culture method used was through explants obtained from three regions of the equine hoof (medial dorsal, dorsal, and lateral dorsal). After the cell isolation and cultivation, the cell culture and its explants were stained with anti-pan cytokeratin (pan-CK) (AE1/AE3), vimentin (V9), p63 (4A4), and Ki-67 (MIB-1) antibodies. Cells were grown to third passage, were positive for pan-CK, p63 and Ki-67, and few cells had vimentin positive expression. As for the explants, the epidermal laminae were not stained for vimentin or Ki-67. However, some cells presented positive pan-CK and p63 expression. This study demonstrated the viability of lamellar explants of equine hooves as a form of isolating keratinocytes in primary cultures, as well as characterized the proliferation ability of such keratinocytes in monolayers.

scholarly journals Recovery of Fusarium oxysporum Fo47 Mutants Affected in Their Biocontrol Activity After Transposition of the Fot1 Element

2002 ◽  
Vol 92 (9) ◽  
pp. 936-945 ◽  
Author(s):  
Sophie Trouvelot ◽  
Chantal Olivain ◽  
Ghislaine Recorbet ◽  
Quirico Migheli ◽  
Claude Alabouvette
Keyword(s):  
Fusarium Oxysporum ◽  
Insertional Mutagenesis ◽  
Wild Type Strain ◽  
Growth Habit ◽  
Antagonistic Activity ◽  
Phenotypic Characterization ◽  
Wild Type ◽  
Biocontrol Activity

To investigate the biocontrol mechanisms by which the antagonistic Fusarium oxysporum strain Fo47 is active against Fusarium wilt, a Fot1 transposon-mediated insertional mutagenesis approach was adopted to generate mutants affected in their antagonistic activity. Ninety strains in which an active Fot1 copy had transposed were identified with a phenotypic assay for excision and tested for their biocontrol activity against F. oxysporum f. sp. lini on flax in greenhouse experiments. Sixteen strains were affected in their capacity to protect flax plants, either positively (more antagonistic than Fo47) or negatively (less antagonistic). The molecular characterization of these mutants confirms the excision of Fot1 and its reinsertion in most of the cases. Moreover, we demonstrate that other transposable elements such as Fot2, impala, and Hop have no transposition activity in the mutant genomes. The phenotypic characterization of these mutants shows that they are affected neither in their in vitro growth habit nor in their competitiveness in soil compared with wild-type strain Fo47. These results show that mutants are not impaired in their saprophytic phase and suggest that the altered biocontrol phenotype should likely be expressed during the interaction with the host plant.

scholarly journals Construction and Phenotypic Characterization of an Auxotrophic Mutant of Mycobacterium tuberculosis Defective in l-Arginine Biosynthesis

2002 ◽  
Vol 70 (6) ◽  
pp. 3080-3084 ◽  
Author(s):  
Bhavna G. Gordhan ◽  
Debbie A. Smith ◽  
Heidi Alderton ◽  
Ruth A. McAdam ◽  
Gregory J. Bancroft ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Auxotrophic Mutant ◽  
Phenotypic Characterization ◽  
Wild Type ◽  
Arginine Biosynthesis ◽  
High Concentrations

ABSTRACT A mutant of Mycobacterium tuberculosis defective in the metabolism of l-arginine was constructed by allelic exchange mutagenesis. The argF mutant strain required exogenous l-arginine for growth in vitro, and in the presence of 0.96 mM l-arginine, it achieved a growth rate and cell density in stationary phase comparable to those of the wild type. The mutant strain was also able to grow in the presence of high concentrations of argininosuccinate, but its auxotrophic phenotype could not be rescued by l-citrulline, suggesting that the ΔargF::hyg mutation exerted a polar effect on the downstream argG gene but not on argH. The mutant strain displayed reduced virulence in immunodeficient SCID mice and was highly attenuated in immunocompetent DBA/2 mice, suggesting that l-arginine availability is restricted in vivo.

scholarly journals Effects of the Ayurved Siriraj Wattana recipe on functional and phenotypic characterization of cytokine-induced killer cells and dendritic cells in vitro

2016 ◽  
Vol 16 (1) ◽  
Author(s):  
Adisak Wongkajornsilp ◽  
Nuntarak Numchaisermsuk ◽  
Khanit Sa-ngiamsuntorn ◽  
Pravit Akarasereenont ◽  
Valla Wamanuttajinda ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Phenotypic Characterization ◽  
Killer Cells

scholarly journals Black soyabean seed coat extract regulates iron metabolism by inhibiting the expression of hepcidin

2014 ◽  
Vol 111 (7) ◽  
pp. 1181-1189 ◽  
Author(s):  
Mingdao Mu ◽  
Aimin Wu ◽  
Peng An ◽  
Xiaoli Du ◽  
Qian Wu ◽  
...  
Keyword(s):  
Chronic Inflammation ◽  
Iron Metabolism ◽  
Seed Coat ◽  
Drug Target ◽  
Fe Deficiency ◽  
Deficiency Anaemia ◽  
Hepcidin Expression ◽  
Fe Homeostasis

Hepcidin, a key regulator of Fe homeostasis, is an ideal drug target for treating patients with Fe disorders such as haemochromatosis, anaemia of chronic inflammation and Fe-deficiency anaemia. However, whether (and how) traditional Chinese black foods (e.g. black soyabeans) target hepcidin and improve Fe-deficiency anaemia remains unclear. Herein, we report that black soyabean seed coat extract (BSSCE) can potently inhibit thein vitroandin vivoexpression of hepcidin. In the present study, in cells treated with 200 μg/ml BSSCE, hepcidin expression was found to be reduced to only 6 % of the control levels (P< 0·01). An AIN-76A diet containing 2 % BSSCE was fed to 8-week-old male C57BL/6 mice for 0, 1, 7, 15 or 30 d; importantly, compared with the day 0 group, the day 7 group exhibited nearly a 50 % decrease in hepatic hepcidin expression (P< 0·01), a 35 % decrease in splenic Fe concentrations (P< 0·05) and a 135 % increase in serum Fe concentrations (P< 0·05). Mechanistically, the effect of BSSCE on hepcidin expression was mediated via a reduction in the phosphorylation levels of mothers against decapentaplegic homolog proteins (Smad)1/5/8. Consequently, the mice in the day 30 group exhibited large increases in erythrocyte counts (111 %v.day 0,P< 0·01), Hb concentrations (109 %,P< 0·01) and haematocrit values (108 %,P< 0·01). In conclusion, these results indicate that black soyabean extract regulates Fe metabolism by inhibiting the expression of hepcidin. This finding can be used to optimise the intervention of patients with hepcidin-related diseases, including Fe-deficiency anaemia.

scholarly journals Single-Cell Transcriptomic Attributes and Unbiased Computational Modeling for the Prediction of Immunomodulatory Potency of Mesenchymal Stromal Cells

2020 ◽  
Author(s):  
Pallab Pradhan ◽  
Paramita Chatterjee ◽  
Hazel Y. Stevens ◽  
Chad Glen ◽  
Camila Medrano-Trochez ◽  
...  
Keyword(s):  
Single Cell ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Computational Models ◽  
Functional Characterization ◽  
Symbolic Regression ◽  
Phenotypic Characterization ◽  
Cell Therapies

ABSTRACTMesenchymal stromal cells (MSCs) are currently being tested in numerous clinical trials as potential cell therapies for the treatment of various diseases and due to their potential immunomodulatory, pro-angiogenic, and regenerative properties. However, variabilities in tissue sources, donors, and manufacturing processes and the lack of defined critical quality attributes (CQAs) and clinically relevant mechanism of action (MoA) pose significant challenges to identify MSC cell therapy products with a predictable therapeutic outcome. This also hinders regulatory considerations and broad clinical translation of MSCs. MSC products are often administered to the patient immediately after thawing from cryopreserved vials (out-of-thaw). However, the qualifying quality-control assays are either performed before cryopreservation, or after culturing the post-thaw cells for 24-48 hours (culture-rescued), none of which represent the out-of-thaw product administered to patients. In this study, we performed a broad functional characterization of out-of-thaw and culture-rescue MSCs from bone marrow (BM-MSCs) and cord tissue (CT-MSCs) using macrophage activation and T cell proliferation-based in vitro potency assays and deep phenotypic characterization using single-cell RNA-sequencing. Using this data, we developed unbiased computational models, specifically symbolic regression (SR) and canonical correlation analysis (CCA) models to predict the immunomodulatory potency of MSCs. Overall, our results suggest that manufacturing conditions (OOT vs. CR) have a strong effect on MSC-function on MSC interactions with macrophages and T cells. Furthermore, single-cell RNA-seq analyses of out-of-thaw BM and CT-MSCs indicate a tissue of origin-dependent variability and heterogeneity in the transcriptome profile. Using symbolic regression modeling we identified specific single-cell transcriptomic attributes of MSCs that predict their immunomodulatory potency. In addition, CCA modeling predicted MSC donors with high or low immunomodulatory potency from their transcriptome profiles. Taken together, our results provide a broad framework for identifying predictive CQAs of MSCs that could ultimately help in better understanding of their MOAs and improved reproducibility and manufacturing control of MSCs.

scholarly journals In VitroResistance Profile of the Hepatitis C Virus NS3 Protease Inhibitor BI 201335

2011 ◽  
Vol 56 (1) ◽  
pp. 569-572 ◽  
Author(s):  
Lisette Lagacé ◽  
Peter W. White ◽  
Christiane Bousquet ◽  
Nathalie Dansereau ◽  
Florence Dô ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protease Inhibitor ◽  
Protease Inhibitors ◽  
Alpha Interferon ◽  
Phenotypic Characterization ◽  
Genotype 1A ◽  
Ns3 Protease

ABSTRACTThein vitroresistance profile of BI 201335 was evaluated through selection and characterization of variants in genotype 1a (GT 1a) and genotype 1b (GT 1b) replicons. NS3 R155K and D168V were the most frequently observed resistant variants. Phenotypic characterization of the mutants revealed shifts in sensitivity specific to BI 201335 that did not alter susceptibility to alpha interferon. In contrast to macrocyclic and covalent protease inhibitors, changes at V36, T54, F43, and Q80 did not confer resistance to BI 201335.

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