90 results on '"Lloyd JA"'
Search Results
2. Alkaloid Distribution in Seeds of Argemone mexicana L. (Papaveraceae)
- Author
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Lloyd Ja Loza-Müller, Felipe Vázquez-Flota, Miriam Monforte-González, and José Ignacio Laines-Hidalgo
- Subjects
food.ingredient ,biology ,Alkaloid ,General Chemistry ,biology.organism_classification ,Argemone mexicana ,Hypocotyl ,chemistry.chemical_compound ,Horticulture ,food ,chemistry ,Seedling ,Germination ,Papaveraceae ,Sanguinarine ,Cotyledon - Abstract
Seeds of Argemone mexicana L. accumulate significative amounts of sanguinarine. The analysis of the distribution of this alkaloid through the tissues of mature seeds revealed that up to 60 % of its contents was found tightly fixed to the different components of the seed external covers where it persisted during seedling germination. Contrastingly, sanguinarine contents in cotyledon accounted for the remaining 40 % and it could have been, at least partially, mobilized to the newly formed hypocotyls during emergence from seeds. Berberine was only detected in immature seeds and in seedlings once cotyledons were totally displayed. These results are discussed as a possible sanguinarine role in the chemical protection during seedlings germination. Resumen. Semillas de Argemone mexicana L. acumulan cantidades elevadas de sanguinarina. Un análisis de la distribución de alcaloides en los diferentes tejidos que componen la semilla reveló que hasta un 60 % del contenido se encontraba fuertemente unido en las capas que forman la cubierta exterior, donde se retuvieron durante la emergencia del hipocótilo. En contraste, los cotiledones presentaron el 40 % restante y parte de ello pudo haber sido movilizado al hipocótilo al emerger. Berberina sólo se observó en semillas inmaduras y en plántulas en desarrollo con los cotiledones desplegados. Estos resultados se discuten en función del posible papel defensivo de la sanguinarina durante la germinación.
- Published
- 2021
3. Large-Area Nanofabrication of Partially Embedded Nanostructures for Enhanced Plasmonic Hot-Carrier Extraction
- Author
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Ng, C, Zeng, P, Lloyd, JA, Chakraborty, D, Roberts, A, Smith, TA, Bach, U, Sader, JE, Davis, TJ, Gomez, DE, Ng, C, Zeng, P, Lloyd, JA, Chakraborty, D, Roberts, A, Smith, TA, Bach, U, Sader, JE, Davis, TJ, and Gomez, DE
- Abstract
When plasmonic nanoparticles are coupled with semiconductors, highly energetic hot carriers can be extracted from the metal-semiconductor interface for various applications in light energy conversion. However, the current quantum yields for hot-electron extraction are generally low. An approach for increasing the extraction efficiency consists of maximizing the contact area between the surface of the metal nanostructure and the electron-accepting material. In this work, we developed an innovative, simple, and scalable fabrication technique that partially embeds colloidal plasmonic nanostructures within a semiconductor TiO2 layer without utilizing any complex top-down nanofabrication method. The successful embedding is confirmed by scanning electron microscopy and atomic force microscopy imaging. Using visible-pump, near-IR probe transient absorption spectroscopy, we also provide evidence that the increase in the surface contact area between the nanostructures and the electron-accepting material leads to an increase in the amount of hot-electron injection into the TiO2 layer.
- Published
- 2019
4. Determinants of Willingness to Pay (WTP) for organic fertiliser: a case of smallholder potato farmers in KwaZulu-Natal, South Africa
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Bhekani Sandile Zondo and Lloyd James S. Baiyegunhi
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extension services ,contingent valuation ,adoption ,land tenure ,multi-stage sampling ,ordered logit ,Agriculture - Abstract
The South African smallholder sector is characterised by relatively low productivity due to persistent deterioration in soil fertility owing to declining organic matter and other essential soil nutrients. Consequently, adoption of sustainable agricultural inputs like organic fertiliser is essential. Although there is sufficient advocacy in the adoption of organic fertiliser, the economic linkage between farmers' socioeconomic factors and willingness to pay (WTP) remains under-explored. This study investigated the determinants of WTP a price premium for organic fertiliser among smallholder potato farmers using primary data collected from 189 smallholder farmers in three municipal areas in KwaZulu-Natal, South Africa, through a multi-stage sampling technique. The data was analysed using the ordered logit model and revealed that marital status, access to extension services, and knowledge of organic fertiliser usage, land ownership, livestock size and distance to the source of organic fertiliser influenced the farmers’ WTP for organic fertiliser. The study found that about 83.6% of the sampled smallholder farmers were willing to pay for organic fertiliser, while about 16.4% of them indicated that they were not willing to pay for organic fertiliser. This result justifies the prospect of commercialisation of organic fertiliser to facilitate the availability of organic fertiliser to those that are willing to pay for it. This study recommends improved access to extension services to enhance technical information dissemination and knowledge of organic fertiliser usage among smallholder farmers. Development of policies that strive to institute security of land tenure among smallholder farmers, which will encourage smallholder farmers WTP is also essential.
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- 2021
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5. Rural households’ social capital and welfare: A case study of Msinga, KwaZulu-Natal, South Africa
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Lloyd James Segun Baiyegunhi
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social capital ,welfare ,rural households ,economic behaviour ,utility maximization ,Agriculture - Abstract
In a household or nations production system, social capital has been recognized as an input having major implications for project design as well as policy development. Using a structured questionnaire, household level data was obtained from a representative sample of 300 rural households in Msinga, KwaZulu-Natal. This study employed the conventional household economic behaviour model under constrained utility maximisation to examine the effect of social capital on the welfare of household, testing the hypothesis that the possession of social capital improves household welfare. The result shows that social capital endowments have a statistically significant positive effect on household welfare, in addition to the some household’s demographic and socio-economic characteristics. The study concluded that, access to social capital among other factors, is very crucial for improved rural household welfare and poverty reduction. It is therefore important for government to have knowledge of existing social groups and networks as this will improve the effectiveness of the present strategies aimed at reducing poverty.
- Published
- 2014
6. Chemistry of the Podocarpaceae. XXXVI. Ring-B conformation of some ring-c aromatic diterpenoids.
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Cambie, RC, Denny, WA, and Lloyd, JA
- Abstract
The ring-B conformations of some 6,7-diacetoxy, 7-acetoxy-6-hydroxy, 6-dehydro, and 7-oxo derivatives of ring-c aromatic diterpenoids have been examined from a study of their n.m.r, spectra.
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- 1972
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7. Preventive evidence into practice (PEP) study: implementation of guidelines to prevent primary vascular disease in general practice protocol for a cluster randomised controlled trial
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Harris Mark F, Lloyd Jane, Litt John, van Driel Mieke, Mazza Danielle, Russell Grant, Smith Jane, Del Mar Chris, Denney-Wilson Elizabeth, Parker Sharon, Krastev Yordanka, Jayasinghe Upali W, Taylor Richard, Zwar Nick, Wilson Jinty, Bolger-Harris Helen, and Waters Justine
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Primary care ,Family medicine ,Guidelines ,Preventive care ,Cardiovascular disease ,Medicine (General) ,R5-920 - Abstract
Abstract Background There are significant gaps in the implementation and uptake of evidence-based guideline recommendations for cardiovascular disease (CVD) and diabetes in Australian general practice. This study protocol describes the methodology for a cluster randomised trial to evaluate the effectiveness of a model that aims to improve the implementation of these guidelines in Australian general practice developed by a collaboration between researchers, non-government organisations, and the profession. Methods We hypothesise that the intervention will alter the behaviour of clinicians and patients resulting in improvements of recording of lifestyle and physiological risk factors (by 20%) and increased adherence to guideline recommendations for: the management of CVD and diabetes risk factors (by 20%); and lifestyle and physiological risk factors of patients at risk (by 5%). Thirty-two general practices will be randomised in a 1:1 allocation to receive either the intervention or continue with usual care, after stratification by state. The intervention will be delivered through: small group education; audit of patient records to determine preventive care; and practice facilitation visits adapted to the needs of the practices. Outcome data will be extracted from electronic medical records and patient questionnaires, and qualitative evaluation from provider and patient interviews. Discussion We plan to disseminate study findings widely and directly inform implementation strategies by governments, professional bodies, and non-government organisations including the partner organisations.
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- 2013
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8. Randomized, controlled, parallel-group prospective study to investigate the clinical effectiveness of early insulin treatment in patients with latent autoimmune diabetes in adults
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Lloyd Janet, Beaverstock Charles, Wareham Kathie, Richards Kez, Cheung Wei-yee, Stephens Jeffrey W, Bain Stephen, Davies Helen, Brophy Sinead, Page Don, Williams Meurig, Russell Ian, and Williams Rhys
- Subjects
Diseases of the endocrine glands. Clinical endocrinology ,RC648-665 - Abstract
Abstract Background Latent autoimmune diabetes in adults [LADA] is a type 1 diabetes that is slowly developing. This means many people are treated as having type 2 diabetes at diagnosis as they are adults who are not immediately insulin dependent. LADA can be distinguished from type 2 diabetes by antibody tests. Patients who are antibody positive have an autoimmune reaction which is similar to that of type 1 diabetes and is not found in type 2 diabetes. We would like to examine the best way of treating LADA in the early phase of the conditions, with tablets (similar to type 2 diabetes) or with insulin (similar to type 1 diabetes). Methods/design This is an open parallel group prospective randomised trial. Participants need to have a GAD antibody test results of 101 WHO units or more and a diagnosis of diabetes not requiring insulin at diagnosis. Participants will need to have been diagnosed within 12 months and not treated with insulin at study entry. They will be randomised to receive either insulin (NovoMix 30) or tablets (diet treated followed by metformin followed by glitazone (with or without metformin) followed by insulin). Primary outcome assessment will be for change in HbA1c and change in fasting C-peptide over 24 months. Secondary outcome measures will include Quality of life, GAD antibody levels, adverse events, inflammatory markers, insulin resistance, and markers of the metabolic syndrome. Discussion This study seeks the best treatment for early LADA in terms of maintaining glycaemic control and maintaining natural insulin production. Trial registration ISRCTN63815121
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- 2008
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9. A Multi-Institutional Description of Processes and Outcomes of Postbaccalaureate Research Education Programs in the Mid-Atlantic Region.
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Wright CF, Kasman LM, Robinson DL, Carey GB, Hall JD, Lloyd JA, Shiang R, Smith EJ, and Wilson KL
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- Adult, Female, Humans, Male, Black or African American statistics & numerical data, Hispanic or Latino statistics & numerical data, Program Evaluation, Schools, Medical organization & administration, South Carolina, United States, Universities, Biomedical Research
- Abstract
Abstract: Outcome data from 6 National Institutes of Health-funded Postbaccalaureate Research Education Programs (PREPs) in the Mid-Atlantic region were combined to give a multi-institutional perspective on their scholars' characteristics and progress through biomedical research training. The institutions hosting these programs were Johns Hopkins University School of Medicine, the Medical University of South Carolina, the University of Maryland School of Medicine, the University of North Carolina at Chapel Hill, Virginia Commonwealth University, and Virginia Polytechnic Institute and State University. The authors summarize the institutional pathways, demographics, undergraduate institutions, and graduate institutions for a total of 384 PREP scholars who completed the programs by June 2021. A total of 228 (59.4%) of these PREP scholars identified as Black or African American, 116 (30.2%) as Hispanic or Latinx, and 269 (70.0%) as female. The authors found that 376 of 384 scholars (97.9%) who started PREP finished their program, 319 of 376 (84.8%) who finished PREP matriculated into PhD or MD/PhD programs, and 284 of 319 (89.0%) who matriculated have obtained their PhD or are successfully making progress toward their PhD., (Copyright © 2023 the Association of American Medical Colleges.)
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- 2024
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10. Self-assembly of spherical and rod-shaped nanoparticles with full positional control.
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Lloyd JA, Liu Y, Ng SH, Thai T, Gómez DE, Widmer-Cooper A, and Bach U
- Abstract
The controlled positioning of spherical gold nanoparticles and gold nanorods upon self-assembly on a substrate is of great interest for the fabrication of tailored plasmonic devices. Here, an electrostatic approach with a sequential two-step assembly protocol is presented as a cost-effective and high-yield alternative to previously presented, more complex proof of concepts. Three different geometries can be separately produced in large quantities relying on electrostatic attraction and repulsion of the charge-carrying building blocks: a single gold nanoparticle at the tip, the side or on top of a gold nanorod. DLVO theory is used to explain the electrostatic assembly strategy. The process is highly efficient and assembly yields between 79% (at the tip) and 94% (for the nanoparticle at the long side of the nanorod) are achieved.
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- 2019
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11. Isomerism control of diethylstilbestrol by metal surface induced O-H cleavage.
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Oh SC, Lloyd JA, Fischer S, Saglam Ö, Papageorgiou AC, Diller K, Duncan DA, Klappenberger F, Allegretti F, Reichert J, and Barth JV
- Abstract
Diethylstilbestrol (DES) is studied on Ag(111) and Cu(111) surfaces using X-ray photoelectron spectroscopy (XPS) and scanning tunnelling microscopy (STM). We find that DES molecules on the silver surface adsorb intact and adopt a trans-conformation. On the more reactive copper surface, O-H bond cleavage results in molecular adsorption in the cis-conformation, thus providing the means of obtaining different adsorption geometries. The difference in isomerism is reflected in the observed self-assemblies which exhibit room-temperature stability.
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- 2018
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12. In Vitro Erythroid Differentiation and Lentiviral Knockdown in Human CD34+ Cells from Umbilical Cord Blood.
- Author
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Kovilakath A, Mohamad S, Hermes F, Wang SZ, Ginder GD, and Lloyd JA
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- Antigens, CD34 metabolism, Cell Culture Techniques, Cell Separation, Erythroid Cells cytology, Erythroid Cells metabolism, Flow Cytometry, Gene Expression, HEK293 Cells, Humans, Transfection, Cell Differentiation genetics, Fetal Blood cytology, Gene Knockdown Techniques, Genetic Vectors genetics, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells metabolism, Lentivirus genetics
- Abstract
Human umbilical cord blood is a rich source of hematopoietic stem and progenitor cells. CD34+ cells in umbilical cord blood are more primitive than those in peripheral blood or bone marrow, and can proliferate at a high rate and differentiate into multiple cell types. In this protocol, a dependable method is described for the isolation of fetal CD34+ cells from umbilical cord blood and expanding these cells in culture. The cells can then be in vitro differentiated along an erythroid pathway, while simultaneously performing knockdown of a gene of choice. The use of lentiviral vectors that express small hairpin RNA (shRNA) is an efficient method to downregulate genes. Flow cytometric analyses are used to enrich for erythroid cells. Using these methods, one can generate in vitro differentiated cells to use for quantitative reverse transcriptase PCR and other purposes.
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- 2018
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13. An Introduction to Erythropoiesis Approaches.
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Lloyd JA
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- Animals, Biomarkers, Cell Differentiation, Cell Line, Transformed, Chromatin Immunoprecipitation, Embryonic Stem Cells cytology, Embryonic Stem Cells metabolism, Erythroid Cells cytology, Erythroid Cells metabolism, Erythroid Precursor Cells cytology, Erythroid Precursor Cells metabolism, Gene Expression Regulation, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells metabolism, Hemoglobins genetics, High-Throughput Nucleotide Sequencing, Humans, Immunophenotyping, Induced Pluripotent Stem Cells cytology, Induced Pluripotent Stem Cells metabolism, Models, Animal, Erythropoiesis
- Abstract
Many experimental models have been used to study erythropoiesis. Even prior to the advent of the genetic manipulation of animal models, erythropoiesis was examined in the mouse, chicken, sheep, goat, and rabbit, among other vertebrates. Erythroid cell lines derived from human blood cancers were also very useful, as they could be genetically manipulated more easily than whole animals. Genetic models in the mouse, zebrafish, and frog have provided a plethora of information advancing our understanding of erythropoiesis, and remain gold standards in the field for studies of hemoglobin switching, and experiments to study authentic blood cell development. Mouse and human embryonic stem (ES) and induced pluripotent (iPS) cells can be differentiated to erythroid cells in culture, though their use is somewhat limited by their propensity to express only the embryonic and fetal globin genes. Some very useful cell lines have been developed by manipulating ES or fetal liver erythroid progenitor cells from knockout mouse models. In recent years, our understanding of erythropoiesis has improved, due to the ability to knock down genes in native human hematopoietic stem and progenitor cells derived from umbilical cord blood or bone marrow, and differentiate them ex vivo to the erythroid lineage. These native cells, and cell lines derived from them, are now providing essential information about human erythropoiesis, which is complementary to that obtained from animal studies. This review provides some perspective about the cell and animal models used to study erythropoiesis over the years.
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- 2018
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14. Comparative profiling of well-defined copper reagents and precursors for the trifluoromethylation of aryl iodides.
- Author
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Kaplan PT, Lloyd JA, Chin MT, and Vicic DA
- Abstract
A number of copper reagents were compared for their effectiveness in trifluoromethylating 4-iodobiphenyl, 4-iodotoluene, and 2-iodotoluene. Yields over time were plotted in order to refine our understanding of each reagent performance, identify any bottlenecks, and provide more insight into the rates of the reactions. Interestingly, differences in reactivity were observed when a well-defined [LCuCF
3 ] complex was employed directly or generated in situ from precursors by published reports. Relative reactivities were also found to highly dependent on the nature of the iodoarenes.- Published
- 2017
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15. A mild thermomechanical process for the enzymatic conversion of radiata pine into fermentable sugars and lignin.
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Suckling ID, Jack MW, Lloyd JA, Murton KD, Newman RH, Stuthridge TR, Torr KM, and Vaidya AA
- Abstract
Background: Conversion of softwoods into sustainable fuels and chemicals is important for parts of the world where softwoods are the dominant forest species. While they have high theoretical sugar yields, softwoods are amongst the most recalcitrant feedstocks for enzymatic processes, typically requiring both more severe pretreatment conditions and higher enzyme doses than needed for other lignocellulosic feedstocks. Although a number of processes have been proposed for converting softwoods into sugars suitable for fuel and chemical production, there is still a need for a high-yielding, industrially scalable and cost-effective conversion route., Results: We summarise work leading to the development of an efficient process for the enzymatic conversion of radiata pine ( Pinus radiata ) into wood sugars. The process involves initial pressurised steaming of wood chips under relatively mild conditions (173 °C for 3-72 min) without added acid catalyst. The steamed chips then pass through a compression screw to squeeze out a pressate rich in solubilised hemicelluloses. The pressed chips are disc-refined and wet ball-milled to produce a substrate which is rapidly saccharified using commercially available enzyme cocktails. Adding 0.1% polyethylene glycol during saccharification was found to be particularly effective with these substrates, reducing enzyme usage to acceptable levels, e.g. 5 FPU/g OD substrate. The pressate is separately hydrolysed using acid, providing additional hemicellulose-derived sugars, for an overall sugar yield of 535 kg/ODT chips (76% of theoretical). The total pretreatment energy input is comparable to other processes, with the additional energy for attrition being balanced by a lower thermal energy requirement. This pretreatment strategy produces substrates with low levels of fermentation inhibitors, so the glucose-rich mainline and pressate syrups can be fermented to ethanol without detoxification. The lignin from the process remains comparatively unmodified, as evident from the level of retained β-ether interunit linkages, providing an opportunity for conversion into saleable co-products., Conclusions: This process is an efficient route for the enzymatic conversion of radiata pine, and potentially other softwoods, into a sugar syrup suitable for conversion into fuels and chemicals. Furthermore, the process uses standard equipment that is largely proven at commercial scale, de-risking process scale-up.
- Published
- 2017
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16. Experimental Lung Injury Reduces Krüppel-like Factor 2 to Increase Endothelial Permeability via Regulation of RAPGEF3-Rac1 Signaling.
- Author
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Huang RT, Wu D, Meliton A, Oh MJ, Krause M, Lloyd JA, Nigdelioglu R, Hamanaka RB, Jain MK, Birukova A, Kress JP, Birukov KG, Mutlu GM, and Fang Y
- Subjects
- Animals, Disease Models, Animal, Guanine Nucleotide Exchange Factors metabolism, Humans, Male, Mice, Mice, Inbred C57BL, Neuropeptides metabolism, Rats, Rats, Sprague-Dawley, rac1 GTP-Binding Protein metabolism, Capillary Permeability physiology, Endothelium, Vascular metabolism, GTP Phosphohydrolases metabolism, Kruppel-Like Transcription Factors metabolism, Respiratory Distress Syndrome metabolism, Signal Transduction physiology
- Abstract
Rationale: Acute respiratory distress syndrome (ARDS) is caused by widespread endothelial barrier disruption and uncontrolled cytokine storm. Genome-wide association studies (GWAS) have linked multiple genes to ARDS. Although mechanosensitive transcription factor Krüppel-like factor 2 (KLF2) is a major regulator of endothelial function, its role in regulating pulmonary vascular integrity in lung injury and ARDS-associated GWAS genes remains poorly understood., Objectives: To examine KLF2 expression in multiple animal models of acute lung injury and further elucidate the KLF2-mediated pathways involved in endothelial barrier disruption and cytokine storm in experimental lung injury., Methods: Animal and in vitro models of acute lung injury were used to characterize KLF2 expression and its downstream effects responding to influenza A virus (A/WSN/33 [H1N1]), tumor necrosis factor-α, LPS, mechanical stretch/ventilation, or microvascular flow. KLF2 manipulation, permeability measurements, small GTPase activity, luciferase assays, chromatin immunoprecipitation assays, and network analyses were used to determine the mechanistic roles of KLF2 in regulating endothelial monolayer integrity, ARDS-associated GWAS genes, and lung pathophysiology., Measurements and Main Results: KLF2 is significantly reduced in several animal models of acute lung injury. Microvascular endothelial KLF2 is significantly induced by capillary flow but reduced by pathologic cyclic stretch and inflammatory stimuli. KLF2 is a novel activator of small GTPase Ras-related C3 botulinum toxin substrate 1 by transcriptionally controlling Rap guanine nucleotide exchange factor 3/exchange factor directly activated by cyclic adenosine monophosphate, which maintains vascular integrity. KLF2 regulates multiple ARDS GWAS genes related to cytokine storm, oxidation, and coagulation in lung microvascular endothelium. KLF2 overexpression ameliorates LPS-induced lung injury in mice., Conclusions: Disruption of endothelial KLF2 results in dysregulation of lung microvascular homeostasis and contributes to lung pathology in ARDS.
- Published
- 2017
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17. Plasmonic Nanolenses: Electrostatic Self-Assembly of Hierarchical Nanoparticle Trimers and Their Response to Optical and Electron Beam Stimuli.
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Lloyd JA, Ng SH, Liu AC, Zhu Y, Chao W, Coenen T, Etheridge J, Gómez DE, and Bach U
- Abstract
Asymmetric nanoparticle trimers composed of particles with increasing diameter act as "plasmonic lenses" and have been predicted to exhibit ultrahigh confinement of electromagnetic energy in the space between the two smallest particles. Here we present an electrostatic self-assembly approach for creating gold nanoparticle trimers with an assembly yield of over 60%. We demonstrate that the trimer assembly leads to characteristic red-shifts and show the localization of the relevant plasmon modes by means of cathodoluminescence and electron energy loss spectroscopy. The results are analyzed in terms of surface plasmon hybridization.
- Published
- 2017
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18. Micromorphological changes and mechanism associated with wet ball milling of Pinus radiata substrate and consequences for saccharification at low enzyme loading.
- Author
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Vaidya AA, Donaldson LA, Newman RH, Suckling ID, Campion SH, Lloyd JA, and Murton KD
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- Cellulase chemistry, Cellulase metabolism, Cellulose analysis, Cellulose chemistry, Hydrolysis, Magnetic Resonance Spectroscopy, Microscopy, Electron, Scanning, Models, Theoretical, Pinus metabolism, Wood metabolism, beta-Glucosidase chemistry, beta-Glucosidase metabolism, Pinus chemistry, Wood chemistry
- Abstract
In this work, substrates prepared from thermo-mechanical treatment of Pinus radiata chips were vibratory ball milled for different times. In subsequent enzymatic hydrolysis, percent glucan conversion passed through a maximum value at a milling time of around 120min and then declined. Scanning electron microscopy revealed breakage of fibers to porous fragments in which lamellae and fibrils were exposed during ball milling. Over-milling caused compression of the porous fragments to compact globular particles with a granular texture, decreasing accessibility to enzymes. Carbon-13 NMR spectroscopy showed partial loss of interior cellulose in crystallites, leveling off once fiber breakage was complete. A mathematical model based on observed micromorphological changes supports ball milling mechanism. At a low enzyme loading of 2FPU/g of substrate and milling time of 120min gave a total monomeric sugar yield of 306g/kg of pulp which is higher than conventional pretreatment method such as steam exploded wood., (Copyright © 2016 Elsevier Ltd. All rights reserved.)
- Published
- 2016
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19. Dynamics of Spatially Confined Bisphenol A Trimers in a Unimolecular Network on Ag(111).
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Lloyd JA, Papageorgiou AC, Fischer S, Oh SC, Saǧlam Ö, Diller K, Duncan DA, Allegretti F, Klappenberger F, Stöhr M, Maurer RJ, Reuter K, Reichert J, and Barth JV
- Abstract
Bisphenol A (BPA) aggregates on Ag(111) shows a polymorphism between two supramolecular motifs leading to formation of distinct networks depending on thermal energy. With rising temperature a dimeric pairing scheme reversibly converts into a trimeric motif, which forms a hexagonal superstructure with complex dynamic characteristics. The trimeric arrangements notably organize spontaneously into a self-assembled one-component array with supramolecular BPA rotors embedded in a two-dimensional stator sublattice. By varying the temperature, the speed of the rotors can be controlled as monitored by direct visualization. A combination of scanning tunneling microscopy and dispersion-corrected density-functional tight-binding (DFTB-vdW(surf)) based molecular modeling reveals the exact atomistic position of each molecule within the assembly as well as the driving force for the formation of the supramolecular rotors.
- Published
- 2016
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20. Krüppel-Like Transcription Factor KLF1 Is Required for Optimal γ- and β-Globin Expression in Human Fetal Erythroblasts.
- Author
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Vinjamur DS, Alhashem YN, Mohamad SF, Amin P, Williams DC Jr, and Lloyd JA
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- Carrier Proteins physiology, Fetal Blood, Gene Knockdown Techniques, Humans, Immunoglobulin Class Switching genetics, Nuclear Proteins physiology, Repressor Proteins, Erythroblasts metabolism, Gene Expression Regulation, Developmental, Kruppel-Like Transcription Factors physiology, beta-Globins genetics, gamma-Globins genetics
- Abstract
In human adult erythroid cells, lower than normal levels of Krüppel-like transcription factor 1 (KLF1) are generally associated with decreased adult β- and increased fetal γ-globin gene expression. KLF1 also regulates BCL11A, a known repressor of adult γ-globin expression. In seeming contrast to the findings in adult cells, lower amounts of KLF1 correlate with both reduced embryonic and reduced fetal β-like globin mRNA in mouse embryonic erythroid cells. The role of KLF1 in primary human fetal erythroid cells, which express both γ- and β-globin mRNA, is less well understood. Therefore, we studied the role of KLF1 in ex vivo differentiated CD34+ umbilical cord blood cells (UCB erythroblasts), representing the fetal milieu. In UCB erythroblasts, KLF1 binds to the β-globin locus control region (LCR), and the β-globin promoter. There is very little KLF1 binding detectable at the γ-globin promoter. Correspondingly, when cultured fetal UCB erythroblasts are subjected to lentiviral KLF1 knockdown, the active histone mark H3K4me3 and RNA pol II recruitment are diminished at the β- but not the γ-globin gene. The amount of KLF1 expression strongly positively correlates with β-globin mRNA and weakly positively correlates with BCL11A mRNA. With modest KLF1 knockdown, mimicking haploinsufficiency, γ-globin mRNA is increased in UCB erythroblasts, as is common in adult cells. However, a threshold level of KLF1 is evidently required, or there is no absolute increase in γ-globin mRNA in UCB erythroblasts. Therefore, the role of KLF1 in γ-globin regulation in fetal erythroblasts is complex, with both positive and negative facets. Furthermore, in UCB erythroblasts, diminished BCL11A is not sufficient to induce γ-globin in the absence of KLF1. These findings have implications for the manipulation of BCL11A and/or KLF1 to induce γ-globin for therapy of the β-hemoglobinopathies.
- Published
- 2016
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21. Surface-Assisted Cyclodehydrogenation; Break the Symmetry, Enhance the Selectivity.
- Author
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Wiengarten A, Lloyd JA, Seufert K, Reichert J, Auwärter W, Han R, Duncan DA, Allegretti F, Fischer S, Oh SC, Sağlam Ö, Jiang L, Vijayaraghavan S, Écija D, Papageorgiou AC, and Barth JV
- Abstract
Selectivity in chemical reactions is a major objective in industrial processes to minimize spurious byproducts and to save scarce resources. In homogeneous catalysis the most important factor which determines selectivity is structural symmetry. However, a transfer of the symmetry concept to heterogeneous catalysis still requires a detailed comprehension of the underlying processes. Here, we investigate a ring-closing reaction in surface-confined meso-substituted porphyrin molecules by scanning tunneling microscopy, temperature-programmed desorption, and computational modeling. The identification of reaction intermediates enables us to analyze the reaction pathway and to conclude that the symmetry of the porphyrin core is of pivotal importance regarding product yields., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2015
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22. Temperature-dependent templated growth of porphine thin films on the (111) facets of copper and silver.
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Diller K, Klappenberger F, Allegretti F, Papageorgiou AC, Fischer S, Duncan DA, Maurer RJ, Lloyd JA, Oh SC, Reuter K, and Barth JV
- Abstract
The templated growth of the basic porphyrin unit, free-base porphine (2H-P), is characterized by means of X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine-structure (NEXAFS) spectroscopy measurements and density functional theory (DFT). The DFT simulations allow the deconvolution of the complex XPS and NEXAFS signatures into contributions originating from five inequivalent carbon atoms, which can be grouped into C-N and C-C bonded species. Polarization-dependent NEXAFS measurements reveal an intriguing organizational behavior: On both Cu(111) and Ag(111), for coverages up to one monolayer, the molecules adsorb undeformed and parallel to the respective metal surface. Upon increasing the coverage, however, the orientation of the molecules in the thin films depends on the growth conditions. Multilayers deposited at low temperatures exhibit a similar average tilting angle (30° relative to the surface plane) on both substrates. Conversely, for multilayers grown at room temperature a markedly different scenario exists. On Cu(111) the film thickness is self-limited to a coverage of approximately two layers, while on Ag(111) multilayers can be grown easily and, in contrast to the bulk 2H-P crystal, the molecules are oriented perpendicular to the surface. This difference in molecular orientation results in a modified line-shape of the C 1s XPS signatures, which depends on the incident photon energy and is explained by comparison with depth-resolved DFT calculations. Simulations of ionization energies for differently stacked molecules show no indication for a packing-induced modification of the multilayer XP spectra, thus indicating that the comparison of single molecule calculations to multilayer data is justified.
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- 2014
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23. Krüppel-like transcription factors KLF1 and KLF2 have unique and coordinate roles in regulating embryonic erythroid precursor maturation.
- Author
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Vinjamur DS, Wade KJ, Mohamad SF, Haar JL, Sawyer ST, and Lloyd JA
- Subjects
- Anemia genetics, Anemia metabolism, Animals, Cell Cycle genetics, Embryo, Mammalian, Gene Expression Regulation, Developmental, Genotype, Kruppel-Like Transcription Factors metabolism, Mice, Mice, Knockout, Phenotype, Cell Differentiation genetics, Erythroid Precursor Cells cytology, Erythroid Precursor Cells metabolism, Erythropoiesis genetics, Kruppel-Like Transcription Factors genetics
- Abstract
The Krüppel-like transcription factors KLF1 and KLF2 are essential for embryonic erythropoiesis. They can partially compensate for each other during mouse development, and coordinately regulate numerous erythroid genes, including the β-like globins. Simultaneous ablation of KLF1 and KLF2 results in earlier embryonic lethality and severe anemia. In this study, we determine that this anemia is caused by a paucity of blood cells, and exacerbated by diminished β-like globin gene expression. The anemia phenotype is dose-dependent, and, interestingly, can be ameliorated by a single copy of the KLF2, but not the KLF1 gene. The roles of KLF1 and KLF2 in maintaining normal peripheral blood cell numbers and globin mRNA amounts are erythroid cell-specific. Mechanistic studies led to the discovery that KLF2 has an essential function in erythroid precursor maintenance. KLF1 can partially compensate for KLF2 in this role, but is uniquely crucial for erythroid precursor proliferation through its regulation of G1- to S-phase cell cycle transition. A more drastic impairment of primitive erythroid colony formation from embryonic progenitor cells occurs with simultaneous loss of KLF1 and KLF2 than with loss of a single factor. KLF1 and KLF2 coordinately regulate several proliferation-associated genes, including Foxm1. Differential expression of FoxM1, in particular, correlates with the observed KLF1 and KLF2 gene dosage effects on anemia. Furthermore, KLF1 binds to the FoxM1 gene promoter in blood cells. Thus KLF1 and KLF2 coordinately regulate embryonic erythroid precursor maturation through the regulation of multiple homeostasis-associated genes, and KLF2 has a novel and essential role in this process., (Copyright© Ferrata Storti Foundation.)
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- 2014
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24. Self-assembly and chemical modifications of bisphenol a on Cu(111): interplay between ordering and thermally activated stepwise deprotonation.
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Fischer S, Papageorgiou AC, Lloyd JA, Oh SC, Diller K, Allegretti F, Klappenberger F, Seitsonen AP, Reichert J, and Barth JV
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- Microscopy, Scanning Tunneling, Photoelectron Spectroscopy, Benzhydryl Compounds chemistry, Copper chemistry, Phenols chemistry, Protons
- Abstract
Bisphenol A (BPA) is a chemical widely used in the synthesis pathway of polycarbonates for the production of many daily used products. Besides other adverse health effects, medical studies have shown that BPA can cause DNA hypomethylation and therefore alters the epigenetic code. In the present work, the reactivity and self-assembly of the molecule was investigated under ultra-high-vacuum conditions on a Cu(111) surface. We show that the surface-confined molecule goes through a series of thermally activated chemical transitions. Scanning tunneling microscopy investigations showed multiple distinct molecular arrangements dependent on the temperature treatment and the formation of polymer-like molecular strings for temperatures above 470 K. X-ray photoelectron spectroscopy measurements revealed the stepwise deprotonation of the hydroxy groups, which allows the molecules to interact strongly with the underlying substrate as well as their neighboring molecules and therefore drive the organization into distinct structural arrangements. On the basis of the combined experimental evidence in conjunction with density functional theory calculations, structural models for the self-assemblies after the thermal treatment were elaborated.
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- 2014
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25. Laser capture microdissection of embryonic cells and preparation of RNA for microarray assays.
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Redmond LC, Pang CJ, Dumur C, Haar JL, and Lloyd JA
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- Animals, Embryo, Mammalian, Mice, Molecular Biology methods, Nucleic Acid Hybridization, Oligonucleotide Array Sequence Analysis methods, RNA isolation & purification, Gene Expression Profiling, Laser Capture Microdissection methods, RNA biosynthesis
- Abstract
In order to compare the global gene expression profiles of different embryonic cell types, it is first necessary to isolate the specific cells of interest. The purpose of this chapter is to provide a step-by-step protocol to perform laser capture microdissection (LCM) on embryo samples and obtain sufficient amounts of high-quality RNA for microarray hybridizations. Using the LCM/microarray strategy on mouse embryo samples has some challenges, because the cells of interest are available in limited quantities. The first step in the protocol is to obtain embryonic tissue, and immediately cryoprotect and freeze it in a cryomold containing Optimal Cutting Temperature freezing media (Sakura Finetek), using a dry ice-isopentane bath. The tissue is then cryosectioned, and the microscope slides are processed to fix, stain, and dehydrate the cells. LCM is employed to isolate specific cell types from the slides, identified under the microscope by virtue of their morphology. Detailed protocols are provided for using the currently available ArcturusXT LCM instrument and CapSure(®) LCM Caps, to which the selected cells adhere upon laser capture. To maintain RNA integrity, upon removing a slide from the final processing step, or attaching the first cells on the LCM cap, LCM is completed within 20 min. The cells are then immediately recovered from the LCM cap using a denaturing solution that stabilizes RNA integrity. RNA is prepared using standard methods, modified for working with small samples. To ensure the validity of the microarray data, the quality of the RNA is assessed using the Agilent bioanalyzer. Only RNA that is of sufficient integrity and quantity is used to perform microarray assays. This chapter provides guidance regarding troubleshooting and optimization to obtain high-quality RNA from cells of limited availability, obtained from embryo samples by LCM.
- Published
- 2014
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26. Improved bioactivity of G-rich triplex-forming oligonucleotides containing modified guanine bases.
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Rogers FA, Lloyd JA, and Tiwari MK
- Subjects
- Animals, Binding Sites, DNA genetics, DNA metabolism, DNA Breaks, Double-Stranded, Gene Targeting, Genes, Reporter, Guanine chemistry, Mice, Mutagenesis, Oligonucleotides genetics, Oligonucleotides metabolism, Potassium chemistry, DNA chemistry, Nucleosides chemistry, Oligonucleotides chemistry, Pyrimidinones chemistry
- Abstract
Triplex structures generated by sequence-specific triplex-forming oligonucleotides (TFOs) have proven to be promising tools for gene targeting strategies. In addition, triplex technology has been highly utilized to study the molecular mechanisms of DNA repair, recombination and mutagenesis. However, triplex formation utilizing guanine-rich oligonucleotides as third strands can be inhibited by potassium-induced self-association resulting in G-quadruplex formation. We report here that guanine-rich TFOs partially substituted with 8-aza-7-deaza-guanine (PPG) have improved target site binding in potassium compared with TFOs containing the natural guanine base. We designed PPG-substituted TFOs to bind to a polypurine sequence in the supFG1 reporter gene. The binding efficiency of PPG-substituted TFOs to the target sequence was analyzed using electrophoresis mobility gel shift assays. We have determined that in the presence of potassium, the non-substituted TFO, AG30 did not bind to its target sequence, however binding was observed with the PPG-substituted AG30 under conditions with up to 140 mM KCl. The PPG-TFOs were able to maintain their ability to induce genomic modifications as measured by an assay for gene-targeted mutagenesis. In addition, these compounds were capable of triplex-induced DNA double strand breaks, which resulted in activation of apoptosis.
- Published
- 2014
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27. The Krüppel-like factor 2 and Krüppel-like factor 4 genes interact to maintain endothelial integrity in mouse embryonic vasculogenesis.
- Author
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Chiplunkar AR, Curtis BC, Eades GL, Kane MS, Fox SJ, Haar JL, and Lloyd JA
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- Animals, Embryo, Mammalian, Gene Expression Regulation, Developmental, Intracranial Hemorrhages embryology, Intracranial Hemorrhages metabolism, Kruppel-Like Factor 4, Kruppel-Like Transcription Factors genetics, Mice, Mice, Knockout, Microscopy, Electron, Morphogenesis, Nitric Oxide Synthase genetics, Nitric Oxide Synthase metabolism, Occludin genetics, Receptor, Fibroblast Growth Factor, Type 2 genetics, Receptor, Fibroblast Growth Factor, Type 2 metabolism, Signal Transduction, Tamoxifen pharmacology, Blood Vessels embryology, Embryonic Development, Endothelium, Vascular embryology, Endothelium, Vascular metabolism, Kruppel-Like Transcription Factors metabolism
- Abstract
Background: Krüppel-like Factor 2 (KLF2) plays an important role in vessel maturation during embryonic development. In adult mice, KLF2 regulates expression of the tight junction protein occludin, which may allow KLF2 to maintain vascular integrity. Adult tamoxifen-inducible Krüppel-like Factor 4 (KLF4) knockout mice have thickened arterial intima following vascular injury. The role of KLF4, and the possible overlapping functions of KLF2 and KLF4, in the developing vasculature are not well-studied., Results: Endothelial breaks are observed in a major vessel, the primary head vein (PHV), in KLF2-/-KLF4-/- embryos at E9.5. KLF2-/-KLF4-/- embryos die by E10.5, which is earlier than either single knockout. Gross hemorrhaging of multiple vessels may be the cause of death. E9.5 KLF2-/-KLF4+/- embryos do not exhibit gross hemorrhaging, but cross-sections display disruptions of the endothelial cell layer of the PHV, and these embryos generally also die by E10.5. Electron micrographs confirm that there are gaps in the PHV endothelial layer in E9.5 KLF2-/-KLF4-/- embryos, and show that the endothelial cells are abnormally bulbous compared to KLF2-/- and wild-type (WT). The amount of endothelial Nitric Oxide Synthase (eNOS) mRNA, which encodes an endothelial regulator, is reduced by 10-fold in E9.5 KLF2-/-KLF4-/- compared to KLF2-/- and WT embryos. VEGFR2, an eNOS inducer, and occludin, a tight junction protein, gene expression are also reduced in E9.5 KLF2-/-KLF4-/- compared to KLF2-/- and WT embryos., Conclusions: This study begins to define the roles of KLF2 and KLF4 in the embryonic development of blood vessels. It indicates that the two genes interact to maintain an intact endothelial layer. KLF2 and KLF4 positively regulate the eNOS, VEGFR2 and occludin genes. Down-regulation of these genes in KLF2-/-KLF4-/- embryos may result in the observed loss of vascular integrity.
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- 2013
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28. Krüppel-like factor 2 is required for normal mouse cardiac development.
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Chiplunkar AR, Lung TK, Alhashem Y, Koppenhaver BA, Salloum FN, Kukreja RC, Haar JL, and Lloyd JA
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- Animals, Embryo, Mammalian cytology, Embryo, Mammalian metabolism, Embryo, Mammalian pathology, Embryo, Mammalian physiopathology, Female, GATA4 Transcription Factor metabolism, Gene Expression Regulation, Developmental, Glycosaminoglycans analysis, Heart physiopathology, Heart Defects, Congenital metabolism, Heart Defects, Congenital pathology, Heart Defects, Congenital physiopathology, Kruppel-Like Transcription Factors metabolism, Male, Mice abnormalities, Mice, Inbred C57BL, Mice, Knockout, Mice, Transgenic, Myocardium cytology, Myocardium metabolism, Myocardium pathology, T-Box Domain Proteins metabolism, Heart embryology, Heart Defects, Congenital genetics, Kruppel-Like Transcription Factors genetics, Mice embryology
- Abstract
Krüppel-like factor 2 (KLF2) is expressed in endothelial cells in the developing heart, particularly in areas of high shear stress, such as the atrioventricular (AV) canal. KLF2 ablation leads to myocardial thinning, high output cardiac failure and death by mouse embryonic day 14.5 (E14.5) in a mixed genetic background. This work identifies an earlier and more fundamental role for KLF2 in mouse cardiac development in FVB/N mice. FVB/N KLF2-/- embryos die earlier, by E11.5. E9.5 FVB/N KLF2-/- hearts have multiple, disorganized cell layers lining the AV cushions, the primordia of the AV valves, rather than the normal single layer. By E10.5, traditional and endothelial-specific FVB/N KLF2-/- AV cushions are hypocellular, suggesting that the cells accumulating at the AV canal have a defect in endothelial to mesenchymal transformation (EMT). E10.5 FVB/N KLF2-/- hearts have reduced glycosaminoglycans in the cardiac jelly, correlating with the reduced EMT. However, the number of mesenchymal cells migrating from FVB/N KLF2-/- AV explants into a collagen matrix is reduced considerably compared to wild-type, suggesting that the EMT defect is not due solely to abnormal cardiac jelly. Echocardiography of E10.5 FVB/N KLF2-/- embryos indicates that they have abnormal heart function compared to wild-type. E10.5 C57BL/6 KLF2-/- hearts have largely normal AV cushions. However, E10.5 FVB/N and C57BL/6 KLF2-/- embryos have a delay in the formation of the atrial septum that is not observed in a defined mixed background. KLF2 ablation results in reduced Sox9, UDP-glucose dehydrogenase (Ugdh), Gata4 and Tbx5 mRNA in FVB/N AV canals. KLF2 binds to the Gata4, Tbx5 and Ugdh promoters in chromatin immunoprecipitation assays, indicating that KLF2 could directly regulate these genes. In conclusion, KLF2-/- heart phenotypes are genetic background-dependent. KLF2 plays a role in EMT through its regulation of important cardiovascular genes.
- Published
- 2013
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29. Kruppel-like factor 1 (KLF1), KLF2, and Myc control a regulatory network essential for embryonic erythropoiesis.
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Pang CJ, Lemsaddek W, Alhashem YN, Bondzi C, Redmond LC, Ah-Son N, Dumur CI, Archer KJ, Haar JL, Lloyd JA, and Trudel M
- Subjects
- Animals, Base Sequence, DNA Primers genetics, Erythroblasts cytology, Erythroblasts metabolism, Female, Gene Expression Profiling, Gene Expression Regulation, Developmental, Kruppel-Like Transcription Factors deficiency, Male, Mice, Mice, Knockout, Pregnancy, Promoter Regions, Genetic, RNA, Messenger genetics, alpha-Globins genetics, beta-Globins genetics, Erythropoiesis genetics, Gene Regulatory Networks, Genes, myc, Kruppel-Like Transcription Factors genetics
- Abstract
The Krüppel-like factor 1 (KLF1) and KLF2 positively regulate embryonic β-globin expression and have additional overlapping roles in embryonic (primitive) erythropoiesis. KLF1(-/-) KLF2(-/-) double knockout mice are anemic at embryonic day 10.5 (E10.5) and die by E11.5, in contrast to single knockouts. To investigate the combined roles of KLF1 and KLF2 in primitive erythropoiesis, expression profiling of E9.5 erythroid cells was performed. A limited number of genes had a significantly decreasing trend of expression in wild-type, KLF1(-/-), and KLF1(-/-) KLF2(-/-) mice. Among these, the gene for Myc (c-Myc) emerged as a central node in the most significant gene network. The expression of the Myc gene is synergistically regulated by KLF1 and KLF2, and both factors bind the Myc promoters. To characterize the role of Myc in primitive erythropoiesis, ablation was performed specifically in mouse embryonic proerythroblast cells. After E9.5, these embryos exhibit an arrest in the normal expansion of circulating red cells and develop anemia, analogous to KLF1(-/-) KLF2(-/-) embryos. In the absence of Myc, circulating erythroid cells do not show the normal increase in α- and β-like globin gene expression but, interestingly, have accelerated erythroid cell maturation between E9.5 and E11.5. This study reveals a novel regulatory network by which KLF1 and KLF2 regulate Myc to control the primitive erythropoietic program.
- Published
- 2012
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30. Transcription factors KLF1 and KLF2 positively regulate embryonic and fetal beta-globin genes through direct promoter binding.
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Alhashem YN, Vinjamur DS, Basu M, Klingmüller U, Gaensler KM, and Lloyd JA
- Subjects
- Animals, Embryo, Mammalian cytology, Erythropoiesis physiology, Humans, Kruppel-Like Transcription Factors genetics, Mice, Mice, Knockout, Protein Binding, beta-Globins genetics, Embryo, Mammalian metabolism, Gene Expression Regulation, Developmental physiology, Genetic Loci physiology, Kruppel-Like Transcription Factors metabolism, Promoter Regions, Genetic physiology, beta-Globins biosynthesis
- Abstract
Krüppel-like factors (KLFs) control cell differentiation and embryonic development. KLF1 (erythroid Krüppel-like factor) plays essential roles in embryonic and adult erythropoiesis. KLF2 is a positive regulator of the mouse and human embryonic β-globin genes. KLF1 and KLF2 have highly homologous zinc finger DNA-binding domains. They have overlapping roles in embryonic erythropoiesis, as demonstrated using single and double KO mouse models. Ablation of the KLF1 or KLF2 gene causes embryonic lethality, but double KO embryos are more anemic and die sooner than either single KO. In this work, a dual human β-globin locus transgenic and KLF knockout mouse model was used. The results demonstrate that the human ε- (embryonic) and γ-globin (fetal) genes are positively regulated by KLF1 and KLF2 in embryos. Conditional KO mouse experiments indicate that the effect of KLF2 on embryonic globin gene regulation is at least partly erythroid cell-autonomous. KLF1 and KLF2 bind directly to the promoters of the human ε- and γ-globin genes, the mouse embryonic Ey- and βh1-globin genes, and also to the β-globin locus control region, as demonstrated by ChIP assays with mouse embryonic blood cells. H3K9Ac and H3K4me3 marks indicate open chromatin and active transcription, respectively. These marks are diminished at the Ey-, βh1-, ε- and γ-globin genes and locus control region in KLF1(-/-) embryos, correlating with reduced gene expression. Therefore, KLF1 and KLF2 positively regulate the embryonic and fetal β-globin genes through direct promoter binding. KLF1 is required for normal histone modifications in the β-globin locus in mouse embryos.
- Published
- 2011
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31. Krüppel-like factor 2 regulated gene expression in mouse embryonic yolk sac erythroid cells.
- Author
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Redmond LC, Dumur CI, Archer KJ, Grayson DR, Haar JL, and Lloyd JA
- Subjects
- Animals, Cell Adhesion Molecules, Neuronal genetics, Cell Line, Extracellular Matrix Proteins genetics, Female, Gene Expression Profiling, Gene Regulatory Networks genetics, Humans, K562 Cells, Male, Mice, Mice, Knockout, Nerve Tissue Proteins genetics, Promoter Regions, Genetic genetics, Reelin Protein, Serine Endopeptidases genetics, Yolk Sac cytology, Erythroid Cells metabolism, Gene Expression Regulation, Developmental, Kruppel-Like Transcription Factors genetics, Kruppel-Like Transcription Factors metabolism, Yolk Sac embryology
- Abstract
KLF2 is a Krüppel-like zinc-finger transcription factor required for blood vessel, lung, T-cell and erythroid development. KLF2-/- mice die by embryonic day 14.5 (E14.5), due to hemorrhaging and heart failure. In KLF2-/- embryos, β-like globin gene expression is reduced, and E10.5 erythroid cells exhibit abnormal morphology. In this study, other genes regulated by KLF2 were identified by comparing E9.5 KLF2-/- and wild-type (WT) yolk sac erythroid precursor cells, using laser capture microdissection and microarray assays. One hundred and ninety-six genes exhibited significant differences in expression between KLF2-/- and WT; eighty-nine of these are downregulated in KLF2-/-. Genes involved in cell migration, differentiation and development are over-represented in the KLF2-regulated gene list. The SOX2 gene, encoding a pluripotency factor, is regulated by KLF2 in both ES and embryonic erythroid cells. Previous work had identified genes with erythroid-enriched expression in the yolk sac. The erythroid-enriched genes reelin, adenylate cyclase 7, cytotoxic T lymphocyte-associated protein 2 alpha, and CD24a antigen are downregulated in KLF2-/- compared to WT and are therefore candidates for controlling primitive erythropoiesis. Each of these genes contains a putative KLF2 binding site(s) in its promoter and/or an intron. Reelin has an established role in neuronal development. Luciferase reporter assays demonstrated that KLF2 directly transactivates the reelin promoter in erythroid cells, validating this approach to identify KLF2 target genes., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
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32. Expression patterns of astrocyte elevated gene-1 (AEG-1) during development of the mouse embryo.
- Author
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Jeon HY, Choi M, Howlett EL, Vozhilla N, Yoo BK, Lloyd JA, Sarkar D, Lee SG, and Fisher PB
- Subjects
- Animals, Astrocytes metabolism, Brain embryology, Brain metabolism, Cell Transformation, Neoplastic, Fluorescent Antibody Technique, Gene Expression Profiling, Hair Follicle embryology, Hair Follicle metabolism, Ki-67 Antigen analysis, Ki-67 Antigen genetics, Liver embryology, Liver metabolism, Membrane Proteins, Mice, Mice, Inbred C57BL, Nerve Tissue Proteins metabolism, Polymerase Chain Reaction, RNA-Binding Proteins, Signal Transduction, Skin embryology, Skin metabolism, Spinal Cord embryology, Spinal Cord metabolism, Cell Differentiation, Cell Proliferation, Embryonic Development, Gene Expression Regulation, Developmental, Nerve Tissue Proteins genetics, Neurogenesis genetics
- Abstract
Expression of astrocyte elevated gene-1 (AEG-1) is elevated in multiple human cancers including brain tumors, neuroblastomas, melanomas, breast cancers, non-small cell lung cancers, liver cancers, prostate cancers, and esophageal cancers. This gene plays crucial roles in tumor cell growth, invasion, angiogenesis and progression to metastasis. In addition, over-expression of AEG-1 protects primary and transformed cells from apoptosis-inducing signals by activating PI3K-Akt signaling pathways. These results suggest that AEG-1 is intimately involved in tumorigenesis and may serve as a potential therapeutic target for various human cancers. However, the normal physiological functions of AEG-1 require clarification. We presently analyzed the expression pattern of AEG-1 during mouse development. AEG-1 was expressed in mid-to-hindbrain, fronto-nasal processes, limbs, and pharyngeal arches in the early developmental period from E8.5 to E9.5. In addition, at stages of E12.5-E18.5 AEG-1 was localized in the brain, and olfactory and skeletal systems suggesting a role in neurogenesis, as well as in skin, including hair follicles, and in the liver, which are organ sites in which AEG-1 has been implicated in tumor development and progression. AEG-1 co-localized with Ki-67, indicating a role in cell proliferation, as previously revealed in tumorigenesis. Taken together, these results suggest that AEG-1 may play a prominent role during normal mouse development in the context of cell proliferation as well as differentiation, and that temporal regulation of AEG-1 expression may be required during specific stages and in specific tissues during development., (Copyright © 2010 Elsevier B.V. All rights reserved.)
- Published
- 2010
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33. KLF8 sets the pace for the cell cycle through interactions with p300 and PCAF.
- Author
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Lloyd JA
- Subjects
- Animals, Cyclin D1 genetics, Cyclin D1 metabolism, G1 Phase, Kruppel-Like Transcription Factors metabolism, Mice, Promoter Regions, Genetic, Transcription Factors chemistry, Transcription Factors genetics, E1A-Associated p300 Protein metabolism, Transcription Factors metabolism, p300-CBP Transcription Factors metabolism
- Published
- 2010
34. Combination atmospheric pressure solids analysis probe and desorption electrospray ionization mass spectrometry ion source.
- Author
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Lloyd JA, Harron AF, and McEwen CN
- Abstract
The atmospheric solids probe analysis (ASAP) probe was investigated as a means for conducting desorption electrospray ionization (DESI) using a commercially available ion source. Solid and liquid samples as well as a raw urine sample were placed on individual melting point tubes and were inserted into either a heated gas stream for ASAP analysis or a charged solvent spray for DESI-like experiments. Samples ranged in polarity, volatility, mass, (100-17,000 Da), and concentration (neat to submicromolar). Mass spectra of multiple samples were obtained within a single acquisition on a short time scale (<30 s per sample). The configuration of the ion source also allowed rapid switching (approximately 1 min) between ASAP, electrospray ionization (ESI), and DESI.
- Published
- 2009
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35. Fragmentation mechanisms of oxidized peptides elucidated by SID, RRKM modeling, and molecular dynamics.
- Author
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Spraggins JM, Lloyd JA, Johnston MV, Laskin J, and Ridge DP
- Subjects
- Computer Simulation, Oxidation-Reduction, Models, Chemical, Oxygen chemistry, Ozone chemistry, Peptides chemistry, Spectrometry, Mass, Electrospray Ionization methods, Spectroscopy, Fourier Transform Infrared methods
- Abstract
The gas-phase fragmentation reactions of singly charged angiotensin II (AngII, DR(+)VYIHPF) and the ozonolysis products AngII+O (DR(+)VY*IHPF), AngII+3O (DR(+)VYIH*PF), and AngII+4O (DR(+)VY*IH*PF) were studied using SID FT-ICR mass spectrometry, RRKM modeling, and molecular dynamics. Oxidation of Tyr (AngII+O) leads to a low-energy charge-remote selective fragmentation channel resulting in the b(4)+O fragment ion. Modification of His (AngII+3O and AngII+4O) leads to a series of new selective dissociation channels. For AngII+3O and AngII+4O, the formation of [MH+3O](+)-45 and [MH+3O](+)-71 are driven by charge-remote processes while it is suggested that b(5) and [MH+3O](+)-88 fragments are a result of charge-directed reactions. Energy-resolved SID experiments and RRKM modeling provide threshold energies and activation entropies for the lowest energy fragmentation channel for each of the parent ions. Fragmentation of the ozonolysis products was found to be controlled by entropic effects. Mechanisms are proposed for each of the new dissociation pathways based on the energies and entropies of activation and parent ion conformations sampled using molecular dynamics.
- Published
- 2009
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36. Reactive uptake of trimethylamine into ammonium nitrate particles.
- Author
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Lloyd JA, Heaton KJ, and Johnston MV
- Subjects
- Aerosols chemistry, Lasers, Mass Spectrometry, Particle Size, Surface Properties, Methylamines chemistry, Nitrates chemistry
- Abstract
The reaction of trimethylamine (TMA) vapor with a polydisperse distribution of ammonium nitrate particles (20-500 nm dia.) was studied in a flow tube reactor with particle analysis by laser desorption (1064 nm) 70 eV electron ionization (EI) in an ion trap time-of-flight (IT-TOF) aerosol mass spectrometer. When the TMA vapor concentration was very high, essentially complete exchange of TMA for ammonia in the particles was achieved. When the TMA vapor concentration was lower ( approximately 500 ppb for a reaction time of 23 s), partial exchange was observed, and the initial reactive uptake coefficient was estimated to be on the order of 2 x 10(-3) at 20% RH. This value suggests that measurable exchange is possible in the atmosphere when particles are exposed to an amine concentration on the order of 1 ppb for a few hours. The effects of particle size, water content, and amine molecular structure on uptake remain to be elucidated.
- Published
- 2009
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37. Erythropoietin-induced phosphorylation/degradation of BIM contributes to survival of erythroid cells.
- Author
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Abutin RM, Chen J, Lung TK, Lloyd JA, Sawyer ST, and Harada H
- Subjects
- Animals, Apoptosis drug effects, Apoptosis Regulatory Proteins genetics, Bcl-2-Like Protein 11, Cell Line, Cell Survival drug effects, Cell Survival genetics, Erythroid Cells cytology, Erythropoietin metabolism, Extracellular Signal-Regulated MAP Kinases genetics, MAP Kinase Signaling System drug effects, Membrane Proteins genetics, Mice, Mice, Knockout, Phosphorylation drug effects, Phosphorylation physiology, Proto-Oncogene Proteins genetics, Apoptosis physiology, Apoptosis Regulatory Proteins metabolism, Erythroid Cells metabolism, Erythropoietin pharmacology, Extracellular Signal-Regulated MAP Kinases metabolism, MAP Kinase Signaling System physiology, Membrane Proteins metabolism, Proto-Oncogene Proteins metabolism
- Abstract
Objective: A proapoptotic BH3-only protein BIM (BCL-2 interacting mediator of cell death) can link cytokine receptor signaling with the apoptotic machinery in hematopoietic cells. We investigated here the role of BIM in erythropoietin (EPO)-mediated survival in erythroid cells., Materials and Methods: We downregulated BIM in EPO-dependent HCD57 erythroid cells with short hairpin RNA (shRNA), and used real-time polymerase chain reaction, Western blots, and flow cytometry to characterize BIM expression and apoptosis. Hematologic analyses of BIM-deficient (Bim(-/-)) mice were conducted., Results: BIM expression increases in primary murine erythroid cells and HCD57 cells deprived of EPO. Whereas Bim mRNA increased less than twofold, BIM protein increased more than 10-fold after EPO withdrawal, suggesting posttranscriptional regulation of BIM. EPO treatment resulted in rapid phosphorylation of BIM at Serine 65 and phosphorylation correlated with degradation of BIM. Inhibition of extracellular signal-regulated kinase (ERK) by a MEK/ERK inhibitor, U0126, blocked both phosphorylation and degradation of BIM, resulting in apoptosis. Treatment with a proteasome inhibitor, MG-132, also blocked degradation of phosphorylated BIM. Downregulation of BIM with the shRNA resulted in HCD57 cells more resistant to apoptosis induced by either EPO withdrawal or ERK inhibition. Although we observed no significant changes in the number of erythrocytes or reticulocytes in the circulation of Bim(-/-) mice, erythroid progenitors from bone marrow in Bim(-/-) mice were reduced in number and more resistant to apoptosis induced by U0126 MEK/ERK inhibitor., Conclusion: EPO protects erythroid cells from apoptosis in part through ERK-mediated phosphorylation followed by proteasomal degradation of BIM.
- Published
- 2009
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38. Left anterior descending coronary aneurysm.
- Author
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Lloyd JA, Weiss ES, and Vricella LA
- Subjects
- Cardiac Tamponade diagnostic imaging, Humans, Male, Tomography, X-Ray Computed, Ultrasonography, Young Adult, Aorta surgery, Coronary Aneurysm diagnostic imaging, Coronary Aneurysm surgery, Coronary Artery Bypass, Saphenous Vein transplantation
- Abstract
A 19-year-old man presented with gradual onset of retrosternal chest pain and hemodynamic instability. Echocardiography and computed tomography showed substantial anterior and posterior pericardial effusion with tamponade. At surgery, a 3-cm ruptured aneurysm of the left anterior descending coronary artery was identified. It was successfully bypassed using a saphenous vein graft anastomosed to the ascending aorta.
- Published
- 2009
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39. Identification of erythroid-enriched gene expression in the mouse embryonic yolk sac using microdissected cells.
- Author
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Redmond LC, Dumur CI, Archer KJ, Haar JL, and Lloyd JA
- Subjects
- Animals, Epithelial Cells metabolism, Gene Expression Profiling, Mice, Microdissection, Oligonucleotide Array Sequence Analysis, Oligonucleotides genetics, Reelin Protein, Reverse Transcriptase Polymerase Chain Reaction, Yolk Sac embryology, Erythroid Cells metabolism, Gene Expression, Gene Regulatory Networks genetics, Yolk Sac metabolism
- Abstract
Little is known about the genes that control the embryonic erythroid program. Laser capture microdissection was used to isolate primitive erythroid precursors and epithelial cells from frozen sections of the embryonic day 9.5 yolk sac. The RNA samples were amplified and labeled for hybridization to Affymetrix GeneChip Mouse Genome 430A 2.0 arrays. Ninety-one genes are expressed significantly higher in erythroid than in epithelial cells. Ingenuity pathway analysis indicates that many of these erythroid-enriched genes cluster in highly significant biological networks. One of these networks contains RBTN2/LMO2, SCL/TAL1, and EKLF/KLF1, three of the very few genes required for primitive erythropoiesis. Quantitative real-time polymerase chain reaction was used to verify that platelet factor 4, reelin, thrombospondin-1, and muscleblind-like 1 mRNA is erythroid-enriched. These genes have established roles in development or differentiation in other systems, and are, therefore, good candidates for regulating primitive erythropoiesis. These results provide a catalog of genes expressed during primitive erythropoiesis.
- Published
- 2008
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40. EKLF and KLF2 have compensatory roles in embryonic beta-globin gene expression and primitive erythropoiesis.
- Author
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Basu P, Lung TK, Lemsaddek W, Sargent TG, Williams DC Jr, Basu M, Redmond LC, Lingrel JB, Haar JL, and Lloyd JA
- Subjects
- Anemia embryology, Anemia genetics, Anemia pathology, Animals, Embryo, Mammalian, Erythrocytes pathology, Female, Fetal Blood metabolism, Gene Expression Regulation, Developmental, Globins metabolism, Male, Mice, Mice, Knockout, Pregnancy, RNA, Messenger metabolism, Yolk Sac pathology, Erythropoiesis genetics, Fetal Blood cytology, Globins genetics, Kruppel-Like Transcription Factors physiology
- Abstract
The Krüppel-like C2/H2 zinc finger transcription factors (KLFs) control development and differentiation. Erythroid Krüppel-like factor (EKLF or KLF1) regulates adult beta-globin gene expression and is necessary for normal definitive erythropoiesis. KLF2 is required for normal embryonic Ey- and betah1-, but not adult betaglobin, gene expression in mice. Both EKLF and KLF2 play roles in primitive erythroid cell development. To investigate potential interactions between these genes, EKLF/KLF2 double-mutant embryos were analyzed. EKLF(-/-)KLF2(-/-) mice appear anemic at embryonic day 10.5 (E10.5) and die before E11.5, whereas single-knockout EKLF(-/-) or KLF2(-/-) embryos are grossly normal at E10.5 and die later than EKLF(-/-)KLF2(-/-) embryos. At E10.5, Ey- and betah1-globin mRNA is greatly reduced in EKLF(-/-)KLF2(-/-), compared with EKLF(-/-) or KLF2(-/-) embryos, consistent with the observed anemia. Light and electron microscopic analyses of E9.5 EKLF(-/-)KLF2(-/-) yolk sacs, and cytospins, indicate that erythroid and endothelial cells are morphologically more abnormal than in either single knockout. EKLF(-/-)KLF2(-/-) erythroid cells are markedly irregularly shaped, suggesting membrane abnormalities. EKLF and KLF2 may have coordinate roles in a common progenitor to erythroid and endothelial cells. The data indicate that EKLF and KLF2 have redundant functions in embryonic beta-like globin gene expression, primitive erythropoiesis, and endothelial development.
- Published
- 2007
- Full Text
- View/download PDF
41. Selective detection of gas-phase TNT by integrated optical waveguide spectrometry using molecularly imprinted sol-gel sensing films.
- Author
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Walker NR, Linman MJ, Timmers MM, Dean SL, Burkett CM, Lloyd JA, Keelor JD, Baughman BM, and Edmiston PL
- Abstract
A chemical sensor was developed to detect the explosive 2,4,6-trinitrotoluene (TNT) utilizing planar integrated optical waveguide (IOW) attenuated total reflection spectrometry. Submicron thick films of organically modified sol-gel polymers were deposited on the waveguide surface as the sensing layer. Sol-gels were molecularly imprinted for TNT using covalently bound template molecules linked to the matrix through 1 or 2 carbamate linkages. Upon chemical cleavage of the template and displacement of the TNT-like pendant groups from the matrix, shape-selective binding sites were created that possess a primary amine group. The amine was used to deprotonate bound TNT yielding an anionic form that absorbs visible light. Binding of TNT and subsequent conversion to the anion results in the attenuation of light propagating through the waveguide, thus creating a spectrophotometric device. Sensitivity can be achieved by taking advantage of the substantial pathlength provided by the use of single mode IOWs. The limit-of-detection to gas-phase TNT was found to be five parts-per-billion (ppbV) in ambient air at a flow rate of 40 mL min(-1) given a 60 s sampling time. The sensor is highly selective for TNT due to the selectivity of binding site recognition of TNT and the subsequent generation of the TNT anion. Response to TNT is not reversible which results in an integrating sensor device which, in theory, can improve the ability to detect small amounts of the explosive if the exposure time is sufficient in length.
- Published
- 2007
- Full Text
- View/download PDF
42. Single-molecule detection of transcription factor binding to DNA in real time: specificity, equilibrium, and kinetic parameters.
- Author
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Nalefski EA, Nebelitsky E, Lloyd JA, and Gullans SR
- Subjects
- Animals, Base Sequence, DNA Primers, Electrophoresis, Polyacrylamide Gel, Fluorescent Dyes, Kinetics, Protein Binding, Spectrometry, Fluorescence, DNA metabolism, DNA-Binding Proteins metabolism, Transcription Factors metabolism
- Abstract
Specificity and temporal control of transcriptional machinery are encoded within sequence-specific transcription factors, of which there are thousands in mammalian genomes. Efforts to completely decipher this code will require an understanding of the DNA binding thermodynamic and kinetic properties displayed by each transcription factor, a daunting task given the current methodologies for measuring these interactions. Here, we present a novel methodology to quantify the binding of proteins to target DNA molecules based on single-molecule detection and real-time counting of individual free and bound fluorescently tagged molecules flowing past a detection device. Using this technology, we measured DNA binding by fluorescently tagged domains of four distinct transcription factors, namely, human early growth response protein Egr-1, vertebrate GATA-1, Drosophila GAGA factor, and lambda bacteriophage Cro repressor. These proteins represent different structural classes (zinc-finger and helix-turn-helix), quaternary states (monomeric and dimeric), and relative affinities (high, intermediate, and low). Specific binding of each protein to its cognate DNA target was demonstrated at low picomolar concentrations. The equilibrium (Kd) and kinetic (kon and koff) constants governing DNA binding by one of these transcription factors, that of Egr-1, were measured using this approach. Kd values obtained from three different types of saturation titrations were reproducible and consistent, yielding values between 10 and 14 pM that, along with the kinetic constants, agree closely with literature values. Because this methodology offers several significant advantages over other existing approaches, namely, real-time determination, requirement for small amounts of reagents, high reproducibility, exquisite sensitivity, and amenability to high-throughput analysis, it is suitable for characterizing DNA-binding proteins as well as other interacting pairs of molecules that can be fluorescently tagged.
- Published
- 2006
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- View/download PDF
43. Single-molecule detection for femtomolar quantification of proteins in heterogeneous immunoassays.
- Author
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Nalefski EA, D'Antoni CM, Ferrell EP, Lloyd JA, Qiu H, Harris JL, and Whitney DH
- Subjects
- Antibodies, Monoclonal, Follicle Stimulating Hormone immunology, Humans, Immunoassay methods, Sensitivity and Specificity, Follicle Stimulating Hormone analysis
- Published
- 2006
- Full Text
- View/download PDF
44. Peptide ozonolysis: product structures and relative reactivities for oxidation of tyrosine and histidine residues.
- Author
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Lloyd JA, Spraggins JM, Johnston MV, and Laskin J
- Subjects
- Computer Simulation, Oxidation-Reduction, Angiotensin II chemistry, Histidine chemistry, Models, Chemical, Models, Molecular, Ozone chemistry, Peptides chemistry, Spectrometry, Mass, Electrospray Ionization methods, Tyrosine chemistry
- Abstract
Angiotensin II (DRVYIHPF) and two analogs, (DRVYIAPA and DRVAIHPA), were used as model systems to study the ozonolysis of peptides containing tyrosine and histidine residues. The ESI mass spectrum of angiotensin II following exposure to ozone showed the formation of adducts containing one, three, and four oxygen atoms. CID and SID spectra of these adducts were consistent with formation of Tyr + O and His + 3O as expected from previous work with amino acids. However, several fragment ions observed in the CID and SID spectra suggested formation of a rather unexpected adduct, Tyr + 3O, and a small amount of the Phe + O adduct. These findings were confirmed by examining two angiotensin analogs. Exposure of DRVYIAPA to ozone resulted in the addition of either one or three oxygen atoms on Tyr, while DRVAIHPA showed only the addition of three oxygen atoms--all on His. Other noteworthy minor oxidation products were observed from these analogs including Tyr + 34 Da, His + 5 Da, His + 34 Da, and His + 82 Da. The reaction rates of the peptides with ozone were found to be similar: second-order rate coefficients are 274 +/- 3, 379 +/- 6, and 439 +/- 34 M(-1) s(-1) for DRVYIAPA, DRVAIHPA, and angiotensin II, respectively. The relative rates indicate (1) an isolated His residue has a slightly greater ozone reactivity than an isolated Tyr residue, and (2) the reaction rates of isolated residues are not additive when both residues are present in the same molecule.
- Published
- 2006
- Full Text
- View/download PDF
45. Isolation of erythroid cells from the mouse embryonic yolk sac by laser capture microdissection and subsequent microarray hybridization.
- Author
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Redmond LC, Haar JL, Giebel ML, Dumur CI, Basu P, Ware JL, and Lloyd JA
- Subjects
- Animals, Embryo, Mammalian cytology, Epithelial Cells cytology, Mice, Oligonucleotide Array Sequence Analysis, RNA, Messenger analysis, Cell Separation methods, Erythroid Precursor Cells cytology, Gene Expression Profiling, Microdissection methods, Yolk Sac cytology
- Abstract
Erythropoietic tissues are complex, containing both erythroid and other cells. The embryonic yolk sac in particular contains primitive erythroid cells in low abundance. Laser capture microdissection (LCM) was performed to isolate erythroid cells, and epithelial cells, from mouse embryonic day 10 (E10) yolk sac. Quantitative RT-PCR was performed to confirm that enriched cell populations were obtained. epsilony- and betaH1-globin mRNAs were enriched in the erythroid compared to the epithelial fraction, and villin mRNA was enriched in the epithelial compared to the erythroid fraction. RNA isolated from the microdissected erythroid cells was of high quality as indicated by capillary electrophoresis. The RNA from the LCM erythroid fraction was linearly amplified with T7 RNA polymerase and hybridized to a Mouse 430A 2.0 Affymetrix array. Forty-eight percent of genes were present in the microarray assays, including low abundance transcripts such as erythroid transcription factors and enzymes involved in heme synthesis. With the LCM/microarray strategy, it will be possible to identify genes that are differentially regulated in native primitive and definitive erythroid cells.
- Published
- 2006
- Full Text
- View/download PDF
46. Identification, characterization, and expression pattern of the chicken EKLF gene.
- Author
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Chervenak AP, Basu P, Shin M, Redmond LC, Sheng G, and Lloyd JA
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Blood Cells metabolism, Brain embryology, Brain metabolism, Chick Embryo, Conserved Sequence, Hematopoiesis, Kruppel-Like Transcription Factors genetics, Molecular Sequence Data, Kruppel-Like Transcription Factors biosynthesis
- Abstract
EKLF/KLF1 was the first of the Krüppel-like factors (KLFs) to be identified in mammals and plays an important role in primitive and definitive erythropoiesis. Here, we identify and characterize EKLF in the chicken (cEKLF). The predicted amino acid sequence of the zinc finger region of cEKLF is at least 87.7% similar to mammalian EKLF proteins and is 98.8% and 95% similar to the EKLF orthologues in Xenopus and zebrafish, respectively. During early embryonic development, cEKLF expression is seen in the posterior primitive streak, which gives rise to hematopoietic cells, and then in the blood islands and in circulating blood cells. cEKLF mRNA is expressed in blood cells but not in brain later in chicken embryonic development. cEKLF mRNA is increased in definitive compared with primitive erythropoiesis. The conserved sequence and expression pattern of cEKLF suggests that its function is similar to its orthologues in mammals, Xenopus, and zebrafish., ((c) 2006 Wiley-Liss, Inc.)
- Published
- 2006
- Full Text
- View/download PDF
47. Sensor for fluorene based on the incorporation of an environmentally sensitive fluorophore proximal to a molecularly imprinted binding site.
- Author
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Carlson CA, Lloyd JA, Dean SL, Walker NR, and Edmiston PL
- Abstract
A fluorescence-based chemical sensor for fluorene was created by molecularly imprinting a sol-gel comprising the bridged silsesquioxane, bis(trimethoxysilylethyl)benzene. The template was covalently bound to the sol-gel matrix using a fluorene analogue functionalized silane. After chemical removal of template via cleavage of a carbamate linkage, an amine group was left that provided an attachment site for the environmentally sensitive fluorescent probe 7-nitrobenz-2-oxa-1,3-diazole (NBD). Fluorene binding was detected by a change in NBD fluorescence intensity induced by a difference in the local polarity around the probe when the recognition site is filled. Such an approach eliminated response to nonspecific binding to the matrix. Sensing films deposited on glass slides were shown to have response times of <60 s and detection limits below 10 parts-per-trillion. Binding experiments demonstrated that the materials had good selectivity for fluorene over close structural analogues including naphthalene, fluoranthene, and anthracene. However, the sensing design is limited by a lack of reversibility following fluorene binding.
- Published
- 2006
- Full Text
- View/download PDF
48. KLF2 is essential for primitive erythropoiesis and regulates the human and murine embryonic beta-like globin genes in vivo.
- Author
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Basu P, Morris PE, Haar JL, Wani MA, Lingrel JB, Gaensler KM, and Lloyd JA
- Subjects
- Animals, Apoptosis, Cytoplasm metabolism, DNA Primers pharmacology, DNA-Binding Proteins metabolism, Embryo, Mammalian ultrastructure, Female, Humans, In Situ Nick-End Labeling, Kruppel-Like Transcription Factors metabolism, Male, Mice, Mice, Knockout, Mice, Transgenic, Microscopy, Electron, RNA, Messenger metabolism, Reverse Transcriptase Polymerase Chain Reaction, Time Factors, Erythropoiesis, Gene Expression Regulation, Globins biosynthesis, Kruppel-Like Transcription Factors physiology
- Abstract
The Krüppel-like factors (KLFs) are a family of C2/H2 zinc finger DNA-binding proteins that are important in controlling developmental programs. Erythroid Krüppel-like factor (EKLF or KLF1) positively regulates the beta-globin gene in definitive erythroid cells. KLF2 (LKLF) is closely related to EKLF and is expressed in erythroid cells. KLF2-/- mice die between embryonic day 12.5 (E12.5) and E14.5, because of severe intraembryonic hemorrhaging. They also display growth retardation and anemia. We investigated the expression of the beta-like globin genes in KLF2 knockout mice. Our results show that KLF2-/- mice have a significant reduction of murine embryonic Ey- and beta h1-globin but not zeta-globin gene expression in the E10.5 yolk sac, compared with wild-type mice. The expression of the adult beta(maj)- and beta(min)-globin genes is unaffected in the fetal livers of E12.5 embryos. In mice carrying the entire human globin locus, KLF2 also regulates the expression of the human embryonic epsilon-globin gene but not the adult beta-globin gene, suggesting that this developmental-stage-specific role is evolutionarily conserved. KLF2 also plays a role in the maturation and/or stability of erythroid cells in the yolk sac. KLF2-/- embryos have a significantly increased number of primitive erythroid cells undergoing apoptotic cell death.
- Published
- 2005
- Full Text
- View/download PDF
49. A functional screen for Krüppel-like factors that regulate the human gamma-globin gene through the CACCC promoter element.
- Author
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Zhang P, Basu P, Redmond LC, Morris PE, Rupon JW, Ginder GD, and Lloyd JA
- Subjects
- Animals, Base Sequence, Cell Cycle Proteins genetics, Cell Cycle Proteins pharmacology, Cell Cycle Proteins physiology, Cell Differentiation genetics, Chickens, DNA-Binding Proteins genetics, DNA-Binding Proteins pharmacology, DNA-Binding Proteins physiology, Drug Evaluation, Preclinical methods, Humans, K562 Cells, Kruppel-Like Factor 4, Kruppel-Like Transcription Factors genetics, Kruppel-Like Transcription Factors pharmacology, Mice, Phylogeny, Promoter Regions, Genetic, RNA, Messenger analysis, Repressor Proteins genetics, Repressor Proteins pharmacology, Repressor Proteins physiology, Transfection, Enhancer Elements, Genetic, Gene Expression Regulation, Globins genetics, Kruppel-Like Transcription Factors physiology
- Abstract
Krüppel-like factors (KLFs) have been systematically screened as potential candidates to regulate human gamma-globin gene expression through its CACCC element. Initially, 21 human proteins that have close sequence similarity to EKLF/KLF1, a known regulator of the human beta-globin gene, were identified. The phylogenetic relationship of these 22 KLF/Sp1 proteins was determined. KLF2/LKLF, KLF3/BKLF, KLF4/GKLF, KLF5/IKLF, KLF8/BKLF3, KLF11/FKLF, KLF12/AP-2rep and KLF13/FKLF2 were chosen for functional screening. Semi-quantitative RT-PCR demonstrated that all eight of these candidates are present in human erythroid cell lines, and that the expression of the KLF2, 4, 5 and 12 mRNAs changed significantly upon erythroid differentiation. Each of the eight KLF mRNAs is expressed in mouse erythroid tissues, throughout development. UV cross-linking assays suggest that multiple erythroid proteins from human cell lines and chicken primary cells interact with the gamma-globin CACCC element. In co-transfection assays in K562 cells, it was demonstrated that KLF2, 5 and 13 positively regulate, and KLF8 negatively regulates, the gamma-globin gene through the CACCC promoter element. The data collectively suggest that multiple KLFs may participate in the regulation of gamma-globin gene expression and that KLF2, 5, 8 and 13 are prime candidates for further study.
- Published
- 2005
- Full Text
- View/download PDF
50. Triplex-forming oligonucleotides as potential tools for modulation of gene expression.
- Author
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Rogers FA, Lloyd JA, and Glazer PM
- Subjects
- Animals, Humans, Mutagens chemistry, Mutagens pharmacology, DNA chemistry, DNA genetics, Gene Expression Regulation genetics, Nucleic Acid Conformation, Oligonucleotides chemistry, Oligonucleotides genetics
- Abstract
Triplex-forming oligonucleotides (TFOs) bind in the major groove of duplex DNA at polypurine/ polypyrimidine stretches in a sequence-specific manner. The binding specificity of TFOs makes them potential candidates for use in directed genome modification. A number of studies have shown that TFOs can introduce permanent changes in a target sequence by stimulating a cell's inherent repair pathways. TFOs have also been demonstrated to inhibit gene expression providing a possible role for these compounds in cancer therapy. This review summarizes the dual roles of TFOs for use in delivering DNA reactive compounds to a specific site in the genome or for introducing permanent changes in the target sequence through the introduction of an altered helical structure. In addition to compiling the ways in which TFOs have been successfully utilized, this review will explore conflicting reports of TFO bioactivity focusing on the variables which affect the efficacy in vitro of TFO mediated genomic modification which in turn may represent the obstacles encountered using TFOs to modulate gene expression in vivo.
- Published
- 2005
- Full Text
- View/download PDF
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