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The African turquoise killifish (Nothobranchius furzeri), the shortest-lived vertebrate that can be bred in captivity, is an emerging model organism for aging research. Here, we describe a multitissue, single-cell gene expression atlas of female and male blood, kidney, liver, and spleen. We annotate 22 cell types, define marker genes, and infer differentiation trajectories. We find pervasive sex-dimorphic gene expression across cell types. Sex-dimorphic genes tend to be linked to lipid metabolism, consistent with clear differences in lipid storage in female vs. male turquoise killifish livers. We use machine learning to predict sex using single-cell gene expression and identify potential markers for molecular sex identity. As a proof of principle, we show that our atlas can be used to deconvolute existing bulk RNA sequencing (RNA-seq) data to obtain accurate estimates of cell type proportions. This atlas can be a resource to the community that could be leveraged to develop cell-type-specific expression in transgenic animals.
Assuntos
Fundulidae , Animais , Feminino , Masculino , Transcriptoma/genética , Caracteres Sexuais , Animais Geneticamente Modificados , EnvelhecimentoRESUMO
The African turquoise killifish (Nothobranchius furzeri), the shortest-lived vertebrate that can be bred in captivity, is an emerging model organism to study vertebrate aging. Here we describe the first multi-tissue, single-cell gene expression atlas of female and male turquoise killifish tissues comprising immune and metabolic cells from the blood, kidney, liver, and spleen. We were able to annotate 22 distinct cell types, define associated marker genes, and infer differentiation trajectories. Using this dataset, we found pervasive sex-dimorphic gene expression across cell types, especially in the liver. Sex-dimorphic genes tended to be involved in processes related to lipid metabolism, and indeed, we observed clear differences in lipid storage in female vs. male turquoise killifish livers. Importantly, we use machine-learning to predict sex using single-cell gene expression in our atlas and identify potential transcriptional markers for molecular sex identity in this species. As proof-of-principle, we show that our atlas can be used to deconvolute existing liver bulk RNA-seq data in this species to obtain accurate estimates of cell type proportions across biological conditions. We believe that this single-cell atlas can be a resource to the community that could notably be leveraged to identify cell type-specific genes for cell type-specific expression in transgenic animals.
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The spastic Han Wistar (sHW) rat serves as a model for human ataxia presenting symptoms of motor deterioration, weight loss, shortened lifespan, and Purkinje neuron loss. Past studies revealed that human neural progenitor cells (NPCs) improved ataxic symptoms at 20 d posttransplantation in sHW rats. In this study, we investigated the fate and longer-term effectiveness of these transplanted NPCs. Rats were placed into four treatment groups: an untreated normal control group (n = 10), an untreated mutant rat control (n = 10), a mutant group that received an injection of dead NPCs (n = 9), and a mutant group that received live NPCs (n = 10). Bilateral cerebellar injections containing 500,000 of either live or dead NPCs were performed on mutant sHW rats at 40 d of age. Motor activity for all mutant rats started to decline in open field testing around day 35. However, at day 45, the live NPC-treated mutants exhibited significant improvements in open field activity. Similar improvements were observed during rotarod testing and weight gain through the completion of the experiments (100 d). Immunohistochemistry revealed few surviving human NPCs in the cerebella of 80- and 100-d-old NPC-treated mutants; while cresyl violet staining revealed that live NPC-treated mutants had significantly more surviving Purkinje neurons compared to mutants that were untreated or received dead NPCs. Direct stereotactic implantation of NPCs alleviated the symptoms of ataxia, acting as a neuroprotectant, supporting future clinical applications of these NPCs in the areas of ataxia as well as other neurodegenerative diseases.
Assuntos
Ataxia/genética , Músculo Esquelético/fisiopatologia , Doenças Neurodegenerativas/genética , Células-Tronco/metabolismo , Animais , Ataxia/patologia , Modelos Animais de Doenças , Humanos , Longevidade , Masculino , Doenças Neurodegenerativas/patologia , Ratos , Ratos WistarRESUMO
Prototypes of on-site automatic photo induced fluorescence detectors of pesticide in natural waters are set up and applied for the determination of the benzoyl- and phenylurea pesticides, namely fluometuron, monolinuron and diflubenzuron. As these pesticides present no native fluorescence the set up system use the photo conversion under UV irradiation of these pesticides into highly fluorescent photoproducts. A first system, called AUTOPIF, (evolution the commercial AQUAPOD system) is develop using a detection via a diode array spectrometer. To improve the sensitivity of the method, a second system, called AUTOPIF+, is developed with a more resolute spectrometer and an intensified CCD camera detection. Analytical applications were carried out in aqueous solution and detected on line with the AUTOPIF and AUTOPIF+ system. The calibration curves are linear over one order of magnitude, and the limits of detection are in the µgâ¯mL-1 range. The analytical performances of these methods for the determination of the three pesticides are satisfactory in comparison to other classical PIF methods published for the determination of phenylurea pesticides in aqueous solutions. Our results show that the AUTOPIF and AUTOPIF+ methods are versatile, sensible and can be easily applied as an alert system to detect pollutant residues in naturals waters over a threshold value.
RESUMO
Electronic absorption and fluorescence spectral properties of new p-substituted-N-phenylpyrroles (N-PhPys), including HOPhPy, MeOPhPy, ThPhPy, PhDPy, DPhDPy, PyPhThThPhPy, and their available, electrosynthesized polymers were investigated. Electronic absorption spectra, fluorescence excitation and emission spectra, fluorescence quantum yields (ΦF) and lifetimes (τF), and other photophysical parameters of these N-PhPy derivatives and their polymers were measured in DMF, DMSO diluted solutions and/or solid state at room temperature. The electronic absorption spectra of N-PhPy derivatives and their polymers included one to several bands, located in the 270-395 nm region, according to the p-phenyl substituent electron-donating effect and conjugated heteroaromatic system length. The fluorescence excitation spectra were characterized by one broad main peak, with, in most cases, one (or more) poorly resolved shoulder (s), appearing in the 270-405 nm region, and their emission spectra were generally constituted of several bands located in the 330-480 nm region. No significant shift of the absorption, fluorescence excitation and emission spectra wavelengths was found upon going from the monomers to the corresponding polymers. ΦF values were high, varying between 0.11 and 0.63, according to the nature of substituents(s) and to the conjugated system extension. Fluorescence decays were mono-exponential for the monomers and poly-exponential for PyPhThThPhPy and for polymers. τF values were relatively short (0.35-5.17 ns), and markedly decreased with the electron-donor character of the phenyl group p-substituent and the conjugated system extension.
Assuntos
Eletroquímica , Elétrons , Modelos Moleculares , Polímeros/química , Polímeros/síntese química , Pirróis/química , Teoria Quântica , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , TemperaturaRESUMO
A new direct laser photo-induced fluorescence high performance liquid chromatography (DL-PIF-HPLC) method is developed for the simultaneous determination of three anilide pesticides, namely carboxin, monalide and propanil. DL-PIF-HPLC uses a tunable Nd:YAG-OPO laser to obtain fluorescent photoproduct(s) and to simultaneously analyze their fluorescence in a short acquisition time with an intensified CCD camera, which improves the selectivity (by choosing the suitable excitation wavelength), increases the sensitivity (due to the high energy of the laser beam) and reduces the time of analysis, relative to the classical PIF methods. However, one of the main drawbacks of PIF methods is the presence of interferences with other compounds, such as other pesticides from the same group yielding similar fluorescent photoproducts, which reduces their selectivity. The analytical interest of DL-PIF-HPLC to avoid these interferences is demonstrated. The DL-PIF spectra, chromatographic conditions and analytical performances of DL-PIF-HPLC are presented for the simultaneous determination of three anilide pesticides. The calibration curves are linear over one order of magnitude and the limits of detection are in the ng mL(-1) range. The new DL-PIF-HPLC system has the advantage to combine the performances of both techniques, DL-PIF and liquid chromatography, and to improve the analysis selectivity.
Assuntos
Carboxina/análise , Cromatografia Líquida de Alta Pressão/métodos , Praguicidas/análise , Propanil/análise , Espectrometria de Fluorescência/métodos , Calibragem , Cromatografia Líquida de Alta Pressão/instrumentação , Lasers , Luz , Limite de Detecção , Metanol , Soluções , Solventes , Espectrometria de Fluorescência/instrumentação , ÁguaRESUMO
A photo-induced fluorescence (PIF) method was developed for the determination of two benzoyl- and phenylurea pesticides, namely diflubenzuron (DFB) and fenuron (FEN). The photoconversion under UV irradiation of both pesticides into strongly fluorescent photoproducts was performed in several media (methanol, ethanol, acetonitrile, pH4 aqueous solution and pH4 water-methanol (30:70, v/v) mixture). PIF parameters were optimized. Analytical figures of merit for the PIF determination of DFB and FEN were satisfactory, with rather wide linear dynamic range (LDR) values of one to two orders of magnitude, relatively low limit of detection (LOD) values of, respectively, 9-24 ng/mL for DFB and 1-28 ng/mL for FEN, and limit of quantification (LOQ) values of, respectively, 30-80 ng/mL for DFB and 4-95 ng/mL for FEN, according to the medium. Relative standard deviation (RSD) values were in the range 1.7-5.6%. PIF was validated by comparing its analytical performances to those of a standard UV absorption spectrophotometric method. The optimized PIF method was applied to the quantitative analysis of both pesticides in various spiked natural water samples collected in a Senegal agricultural area by the standard addition procedure prior to extraction steps in dichloromethane, with satisfactory mean recovery percentage values (97.0-105.3 for DFB and 98.3-102.8% for FEN). An interference study of foreign species, including pesticides and inorganic ions, likely to be present in natural waters, was also carried out.
RESUMO
A direct Laser Photo-Induced Fluorescence (DL-PIF) method is developed for the determination of two phenylurea pesticides, namely fenuron and diflubenzuron. The DL-PIF method uses a tunable Nd:YAG-OPO Laser to obtain the photoproduct(s) and to simultaneously analyse their fluorescence in a short acquisition time on an intensified CCD camera. Compared to classical PIF methods, the use of a tunable laser improves the selectivity (by choosing the suitable excitation wavelength), increases the sensitivity (due to the high energy of the beam) and also reduces the time of analysis. The analytical performances of this method for the determination of both pesticides are satisfactory in comparison to other classical PIF methods published for the determination of phenylurea pesticides. The calibration curves were linear over one order of magnitude and the limits of detection were in the ng mL(-1) range. Satisfactory recoveries were obtained in the analysis of both pesticides in river and sea water spiked samples.
Assuntos
Diflubenzuron/análise , Água Doce/química , Inseticidas/análise , Compostos de Fenilureia/análise , Água do Mar/química , Poluentes Químicos da Água/análise , Calibragem , Humanos , Lasers de Estado Sólido , Luz , Processos Fotoquímicos , Sensibilidade e Especificidade , Espectrometria de Fluorescência/instrumentação , Espectrometria de Fluorescência/métodosRESUMO
Flow injection analysis combined with photo-induced fluorescence (FIA-PIF) has been applied for the determination of α-cypermethrin pesticide residues in Senegalese natural waters, using organic solutions and cyclodextrin (ß-cyclodextrin and 2-hydroxypropyl-ß-cyclodextrin) aqueous media. The α-cypermethrin insecticide has a very weak natural fluorescence, but it is converted into strongly fluorescent photoproduct(s) by UV irradiation. Cyclodextrins were found to enhance the PIF signal. FIA parameters, including mobile phase flow rate, injected volume, and reactor length, were optimized. Analytical performances of the FIA-PIF method for the determination of α-cypermethrin were satisfactory, with concentration linear dynamic ranges over one to two orders of magnitude and with rather low limits of detection and limits of quantification, in the ng mL(-1) range, and relative standard deviations comprised between 1.2% and 3.8%. Application of FIA-PIF for the analysis of fortified natural water samples collected from Senegal yielded good recovery values (84-112%). Because of its high sampling rate, the FIA-PIF method constitutes a rapid analytical tool, useful for quantification of α-cypermethrin residues in natural waters.
Assuntos
Análise de Injeção de Fluxo/métodos , Resíduos de Praguicidas/química , Fotoquímica/métodos , Piretrinas/química , Poluentes Químicos da Água/química , Fluorescência , Processos Fotoquímicos , Fotoquímica/instrumentação , Raios UltravioletaRESUMO
Sulfamethoxazole (SMX) is a synthetic antibiotic widely applied as a bacteriostatic drug to treat a number of diseases. SMX can persist in the environment for long periods of time because of its low biodegradability, which may result in various, direct and indirect, toxicological effects on the environment and on human health. Therefore, we have developed the electrochemical advanced oxidation process (AOP) "electro-Fenton" to degrade SMX in aqueous media. In this work, a detailed study of the evolution of toxicity of SMX and its degradation products in aqueous solutions, during treatment by the electro-Fenton AOP, is described, using the bioluminescence Microtox® method, based on the inhibition of luminescence of marine bacteria Vibrio fischeri. Samples were collected at various electrolysis times and analyzed by HPLC for quantifying the evolution of the degradation products, and their toxicity was measured by the Microtox® method. Our results demonstrated that the toxicity of SMX aqueous solutions varied considerably with the electrolysis time and the applied current intensity. This phenomenon could be explained by the formation and disappearance of several degradation products, including cyclic and/or aromatic intermediates, and short-chain acid carboxylic acids, having a toxicity different of the initial antibiotic. The curves of the % of bacterial luminescence inhibition vs. electrolysis time, corresponding to the evolution of the toxicity of the formed degradation products, were investigated and tentatively interpreted.
Assuntos
Aliivibrio fischeri/efeitos dos fármacos , Antibacterianos/toxicidade , Bioensaio/métodos , Técnicas Eletroquímicas/métodos , Medições Luminescentes/métodos , Sulfametoxazol/toxicidade , Poluentes Químicos da Água/toxicidade , Aliivibrio fischeri/química , Aliivibrio fischeri/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Peróxido de Hidrogênio/química , Ferro/química , Cinética , Oxirredução , Sulfametoxazol/química , Sulfametoxazol/metabolismo , Poluentes Químicos da Água/química , Poluentes Químicos da Água/metabolismoRESUMO
A detailed toxicological study on several pesticides, including chlorothalonil, cyprodynil, dichlobénil, pendimethaline, trifluraline, and alpha-endosulfan, present at trace levels in air and total atmospheric precipitations of Paris is presented. The pesticides contained in the atmospheric samples, collected during sampling campaigns in February-March 2007, are identified and quantified by a high-performance liquid chromatographic (HPLC)-UV detection method. The toxicity measurements are performed by means of the Microtox bioluminescence method, based on the evaluation of the bioluminescence inhibition of the Vibrio fischeri marine bacteria at two exposure times to the pesticide solutions. The specific toxicity, corresponding to the particular toxicity of the compound under study and represented by the EC(50) parameter, is determined for these pesticides. Also, the global toxicity, which is the toxicity of all micro-pollutants present in the sample under study, is estimated for the extracts of air and atmospheric precipitation (rainwater) samples. The specific toxicities strongly vary with the nature of the pesticide, the EC(50) parameter values being comprised between 0.17 and 0.83 mg/mL and 0.15 and 0.66 mg/mL, respectively, for exposure times of 5 and 15 min. The importance of the atmospheric samples' global toxicity and the respective contribution of the toxic potency of the various pesticides contained in these samples are discussed.
Assuntos
Poluentes Atmosféricos/análise , Poluentes Atmosféricos/toxicidade , Aliivibrio fischeri/efeitos dos fármacos , Monitoramento Ambiental/métodos , Medições Luminescentes/métodos , Praguicidas/toxicidade , Chuva , Ar , Compostos de Anilina/análise , Compostos de Anilina/toxicidade , Cromatografia Líquida de Alta Pressão , Endossulfano/análise , Endossulfano/toxicidade , Nitrilas/análise , Nitrilas/toxicidade , Paris , Praguicidas/análise , Sensibilidade e Especificidade , Espectrofotometria Ultravioleta , Trifluralina/análise , Trifluralina/toxicidadeRESUMO
The use of organized supramolecular systems-including micellar media and cyclodextrin inclusion complexes-combined with luminescence techniques in the study and determination of compounds and elements of environmental interest from 1990 to 2005 is reviewed. Analyses of environmental samples performed using fluorescence, photochemically induced fluorescence and phosphorescence spectroscopy as well as liquid chromatography, capillary electrophoresis and flow injection with luminescence detection in the presence of these organized media are described in detail.
RESUMO
Twenty-two pesticides and metabolites selected on the basis of a regional priority list, were surveyed in surface river waters by high performance liquid chromatography coupled in tandem with UV diode array detection and mass spectrometry, after an off-line pre-concentration step. Pesticide concentrations ranged between 0.07 and 4.8 microg/l according to the compound and sampling period. Analytical results were linked to the environmental risk of pesticides, evaluated by their system investigation of risk by integration of score (SIRIS) rank.
Assuntos
Substâncias Perigosas/análise , Resíduos de Praguicidas/análise , Rios/química , Cromatografia Líquida de Alta Pressão , França , Espectrometria de Massas , Raios UltravioletaRESUMO
For moderately hydrophobic compounds such as most pesticides adsorption on colloids (<0.2 microm) may play a key role in pesticide mobility as well as in their degradation by chemical and microbiological processes. However, until now, pesticide-organic colloid interactions are poorly understood. Quantitative data for sorption equilibria on colloids of two series of herbicides including triazines (atrazine, simazine, terbutylazine, prometryne, desethylatrazine, and desisopropylatrazine) and phenylureas (isoproturon, linuron, neburon, and diuron) sampled in the Seine river (urban zone) and the Marne river (agricultural zone) are presented. Partition coefficient of herbicides on colloids (K(com)), were evaluated by solid-phase extraction coupled with high-performance liquid chromatography-UV diode-array detection (SPE-HPLC-UV/DAD). In the case of triazines a satisfactory log-log correlation was found between K(com) and octanol-water coefficient (K(ow)) values. Phenylureas did not obey this correlation, with K(com) values being about two times higher than those of triazines. The existence of two distinct types of adsorption behaviour on colloids partly explains the different occurrence of triazines and phenylureas in surface waters.
Assuntos
Coloides/química , Herbicidas/química , Compostos de Fenilureia/química , Triazinas , Poluentes Químicos da Água/análise , Adsorção , França , Água Doce/análise , Interações Hidrofóbicas e Hidrofílicas , Modelos QuímicosRESUMO
A study on using non-parametric statistical methods was carried out to calculate the binding constant of an inclusion complex and to estimate its associated uncertainty. First, a correct evaluation of the stoichiometry was carried out in order to ensure an accurate determination of the binding constant. For this purpose, the modified Benesi-Hildelbrand method had been previously applied. Then, four statistical methods (three non-parametric methods: two bootstrap approaches, the jackknife method and a parametric one: Fieller's theorem) were employed in order to compute the binding constant. The results obtained from applying these methods and the combination of the methods: jackknife after bootstrap and bootstrap after jackknife were compared. The best results in terms of accuracy were obtained from the application of a bootstrap method: the resampling residuals approach. These procedures were applied to the inclusion complex 2-hydroxil-propyl-beta-cyclodextrin-2,4-dichloro-phenoxyacetic, which shows photochemically-induced fluorescence.
Assuntos
Ciclodextrinas/química , Estatísticas não Paramétricas , Sítios de Ligação , Fluorescência , Cinética , Praguicidas/química , Fotoquímica , SoluçõesRESUMO
In this paper a sensitive and simple method for the resolution of mixtures of chlorophenoxyacid herbicides using photochemical derivatization induced fluorescence has been described. These compounds do not show any fluorescence, hence photolysed to induce fluorescence after direct irradiation with ultraviolet light in presence of a cationic surfactant (cetyltrimethylammonium chloride). Critical variables such as the surfactant concentration and the irradiation time have been optimised for each compound using Sequential Response Surface Methodology (SRSM) by applying Doehlert designs in order to obtain maximum fluorescence intensity. The difference shown between the optimised irradiation times for the formation of the photoproducts allowed us to propose a time-resolved photoactivation method, for the simultaneous determination of binary mixtures, based on the use of different linear calibration curves established at various irradiation times depending on the mixture to be resolved. Satisfactory recoveries were obtained in the analysis of several mixtures of these herbicides at different ratios in spiked waters.
RESUMO
The effects of several biologically important inorganic salts, including NaCl, NaI, NaBr, KCl, MgCl2, MgSO4 and CaCl2 on the electronic absorption and fluorescence spectra of Merocyanine 540 (MC-540) have been investigated in aqueous media at 25 degrees C. Depending on both the MC-540 concentration and the nature of salt, a new absorption band appears at about 515 nm, above the critical salt concentration (CSC), corresponding to salt-induced MC-540 aggregation. Several types of MC-540 fluorescence quenching by the salts are observed, according to their cationic charge and the nature of anion: in the case of monovalent ions (Na+, K+), a non-linear Stern-Volmer behaviour is observed, indicating variable contributions of dynamic and static quenching mechanisms, whereas for divalent alkaline-earth (Mg2+, Ca2+) ions, linear Stern-Volmer relationships are obtained. Using these results, an analytical quenchofluorimetric approach is proposed for the determination of magnesium ions.
Assuntos
Pirimidinonas/química , Sais/química , Espectrometria de Fluorescência/métodos , Ânions , Cálcio/química , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Magnésio/química , Sulfato de Magnésio/química , Modelos Químicos , Potássio/química , Sais/farmacologia , Sódio/química , Cloreto de Sódio/química , TemperaturaRESUMO
First-derivative photochemically induced spectrofluorimetry (PIF-1D) is applied to the simultaneous determination of binary mixtures of 4 sulfonylurea herbicides in aqueous micellar samples. Synthetic binary mixtures of sulfometuronmethyl with chlorsulfuron, metsulfuron-methyl, and 3-rimsulfuron, respectively, are well resolved by using the zero-crossing point procedure. PIF-1D allows the determination of binary mixtures of these herbicides with linear dynamic ranges over about 2 orders of magnitude, limits of detection between 0.5 and 52 ng/mL, and relative standard deviations within 0.3-2.9%. Application to the determination of binary mixtures of these herbicides in spiked tap water samples yielded satisfactory recoveries (90-117%).
Assuntos
Herbicidas/análise , Espectrometria de Fluorescência/métodos , Poluentes Químicos da Água/análise , Sulfonatos de Arila/análise , Fotoquímica , Piridinas/análise , Sulfonamidas/análise , Compostos de Sulfonilureia/análise , Triazinas/análiseRESUMO
In this paper, a combination of a flow injection analysis (FIA) system with micellar-enhanced photochemically induced fluorescence (MEPIF) detection is presented as a powerful alternative for the rapid and sensitive analysis of chlorophenoxyacid herbicides. These compounds do not show native fluorescence but they can be photolysed into strongly fluorescence photoproducts after direct irradiation with ultraviolet light. The use of a cationic surfactant such as cetyltrimethylammonium chloride (CTAC) provides a considerable enhancement of photochemically induced fluorescence intensity and the nature of the technique allows a possible and easy adaptation to a FIA system. In this sense, parameters related to the nature of the analytical signal (pH, irradiation times, surfactant concentration) and to the FIA manifold (injection volume, flow rate and reactor length) have been optimised. Linear calibration graphs, with three replicates for each concentration value were established in the range of 0.2-5.0 mug ml(-1) for 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.1-5.0 mug ml(-1) for Mecoprop (MCPP). The IUPAC detection limits were 73.2 and 33.5 ng ml(-1) for 2,4-D and MCPP, respectively. Satisfactory recoveries were obtained in the analysis of these herbicides in spiked waters.
RESUMO
The electronic absorption and fluorescence excitation and emission spectra of poly(3-methoxythiophene) (PMOT), a soluble, electroactive oligomer mainly constituted of hexamer, were investigated in dimethylsulfoxide at room temperature. The study of PMOT photophysical properties suggests the existence of important pi-electronic delocalization in the oxidized and reduced form of the oligomer. A significant fluorescence quenching of PMOT takes place in the presence of various quenchers such as dimethylterephthalate, potassium iodide and thallium acetate. Quenching mechanisms implying a quencher-induced intersystem-crossing step are proposed. Modified Stern-Volmer relationships were obtained with large quenching bimolecular rate constants (2.7 x 10(9)-6.1 x 10(11) l mol(-1) s(-1)), which suggests electronic energy migration throughout the repeat units of the PMOT hexamer.