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Here, we present the proof-of-concept of a lateral flow assay (LFA) that is capable of detecting small-molecule targets in a noncompetitive manner by deploying a sandwich-type format based on the aptamer kissing complex (AKC) strategy. A fluorescently labeled hairpin aptamer served as the signaling agent, while a specific RNA hairpin grafted onto the strip served as the capture element. The hairpin aptamer switched from an unfolded to a folded form in the presence of the target, resulting in kissing interactions between the loops of the reporter and the capture agents. This design triggered a target-dependent fluorescent signal at the test line. The AKC-based LFA was developed for the detection of adenosine, achieving a detection limit in the micromolar range. The assay revealed the presence of the same analyte in urine. The method also proved effective with another small molecule (theophylline). We believe that the AKC-based LFA approach could overcome many of the shortcomings associated with conventional signal-off methods and competitive processes.
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Adenosina , Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Aptâmeros de Nucleotídeos/química , Adenosina/análise , Adenosina/urina , Técnicas Biossensoriais/métodos , Humanos , Teofilina/análise , Teofilina/urina , Limite de Detecção , Corantes Fluorescentes/químicaRESUMO
BACKGROUND: Endometriosis is a chronic disease characterized by growth of endometrial tissue outside the uterine cavity which could affect 200 million women (The term "woman" is used for convenience. Individuals gendered as man or as nonbinary can also suffer from this disease) worldwide. One of the most common symptoms of endometriosis is pelvic chronic pain associated with fatigue. This pain can cause psychological distress and interpersonal difficulties. As for several chronic diseases, adapted physical activity could help to manage the physical and psychological symptoms. The present study will investigate the effects of a videoconference-based adapted physical activity combined with endometriosis-based education program on quality of life, pain, fatigue, and other psychological symptoms and on physical activity. METHODS: This multicentric randomized-controlled trial will propose to 200 patients with endometriosis to be part of a trial which includes a 6-month program with 45 min to more than 120 min a week of adapted physical activity and/or 12 sessions of endometriosis-based education program. Effects of the program will be compared to a control group in which patients will be placed on a waiting list. All participants will be followed up 3 and 6 months after the intervention. None of the participants will be blind to the allocated trial arm. The primary outcome measure will be quality of life. Secondary outcomes will include endometriosis-related perceived pain, fatigue, physical activity, and also self-image, stereotypes, motivational variables, perceived support, kinesiophobia, basic psychological need related to physical activity, and physical activity barriers. General linear models and multilevel models will be performed. Predictor, moderator, and mediator variables will be investigated. DISCUSSION: This study is one of the first trials to test the effects of a combined adapted physical activity and education program for improving endometriosis symptoms and physical activity. The results will help to improve care for patients with endometriosis. TRIAL REGISTRATION: ClinicalTrials.gov, NCT05831735 . Date of registration: April 25, 2023.
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Endometriose , Qualidade de Vida , Masculino , Humanos , Feminino , Endometriose/diagnóstico , Endometriose/terapia , Endometriose/complicações , Exercício Físico , Dor Pélvica/etiologia , Fadiga , Comunicação por Videoconferência , Terapia por Exercício/efeitos adversos , Ensaios Clínicos Controlados Aleatórios como Assunto , Estudos Multicêntricos como AssuntoRESUMO
Iron is an essential element to the well functionning of the organism and requires careful maintenance of its homeostasis. This is mainly due to hepcidin, a hormone secreted by the liver that controls the flow of iron within the body. It has a hyposideremic action by reducing the expression of ferroportin, the only protein known to this day, which can export iron into the extracellular environment. This has the effect of decreasing intestinal absorption and increasing intracellular retention, especially in macrophages. Hepcidin is stimulated by elevation of iron and inflammation while activation of erythropoiesis inhibits it. Understanding its regulation allows a better understanding of the pathophysiological mechanisms of overload diseases and iron deficiency. Therefore, hepcidin analysis is interesting for the exploration of iron homeostasis. In combination with other biological parameters of iron status, it is possible to find better instructions for therapeutic managements of iron metabolism diseases. Thus, an assaying method by coupling liquid chromatography and tandem mass spectrometry has been implemented at Grenoble University Hospital and the analytical performances of this assay met the laboratory requirements in terms of reliability of the analysis.
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Hepcidinas , Ferro , Humanos , Hepcidinas/metabolismo , Homeostase/fisiologia , Fígado/metabolismo , Reprodutibilidade dos TestesRESUMO
BACKGROUND: In vitro and toxicological studies have shown that non-persistent environmental chemicals can perturb thyroid hormone homeostasis. Epidemiological studies with improved exposure assessment (i.e., repeated urine samples) are needed to evaluate effects of these compounds, individually or as a mixture, in humans. We studied the associations between prenatal exposure to non-persistent environmental chemicals and neonatal thyroid hormones. METHODS: The study population consisted of 442 mother-child pairs from the French SEPAGES mother-child cohort recruited between July 2014 and July 2017. For each participant, four parabens, five bisphenols, triclosan, triclocarban, benzophenone-3 as well as metabolites of phthalates and of di(isononyl)cyclohexane-1,2-dicarboxylate were assessed in two pools of repeated urine samples (median: 21 spot urines per pool), collected in the 2nd and 3rd trimesters of pregnancy, respectively. Thyroid stimulating hormone (TSH) and total thyroxine (T4) levels were determined in newborns from a heel-prick blood spot. Maternal iodine and selenium were assessed in urine and serum, respectively. Adjusted linear regression (uni-pollutant model) and Bayesian Kernel Machine Regression (BKMR, mixture model) were applied to study overall and sex-stratified associations between chemicals and hormone concentrations. RESULTS: Interaction with child sex was detected for several compounds. Triclosan, three parabens, and one phthalate metabolite (OH-MPHP) were negatively associated with T4 among girls in the uni-pollutant model. BKMR also suggested a negative association between the mixture and T4 in girls, whereas in boys the association was positive. The mixture was not linked to TSH levels, and for this hormone the uni-pollutant model revealed associations with only a few compounds. CONCLUSION: Our study, based on repeated urine samples to assess exposure, showed that prenatal exposure to some phenols and phthalates disturb thyroid hormone homeostasis at birth. Furthermore, both uni-pollutant and mixture models, suggested effect modification by child sex, while, to date underlying mechanisms for such sex-differences are not well understood.
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Poluentes Ambientais , Ácidos Ftálicos , Efeitos Tardios da Exposição Pré-Natal , Triclosan , Masculino , Gravidez , Feminino , Humanos , Recém-Nascido , Glândula Tireoide , Parabenos/análise , Triclosan/toxicidade , Teorema de Bayes , Hormônios Tireóideos , Hormônios , Poluentes Ambientais/urina , Tireotropina , Ácidos Ftálicos/toxicidade , Ácidos Ftálicos/urina , Exposição Ambiental/efeitos adversosRESUMO
Herein, we report a novel approach for the design of a colorimetric aptasensor, relying on a Dye Salt Aggregation-based Colorimetric Oligonucleotide assay (DYSACO assay). This method is based on the use of an intercalating agent, Nile Blue (NB), whose aggregation capacities (and thus modification of its absorption spectrum) are drastically amplified by adding salts to the working solution. The presence of an aptamer could protect NB from such aggregation process due to its intercalation into double-stranded DNA and/or interaction with nucleobases. In response to the addition of the specific ligand, the competition between NB and the target for binding to the aptamer occurs, resulting in an increase in the dye salt aggregation and then in the blue-to-blank color change of the solution. The proof-of-principle was demonstrated by employing the anti-l-tyrosinamide aptamer and the assay was successfully applied to the trace enantiomer detection, allowing the detection of an enantiomeric impurity down to approximately 2% in a non-racemic sample. Through a reversed mechanism based on the increased capture of NB by DNA upon analyte binding, the sensing platform was further demonstrated for the Hg(II) detection. Water samples of different origin were spiked with Hg(II) analyte at final range concentrations comprised between (0.5-15 µM). An excellent overall recovery of 122 ± 14%; 105 ± 14%; 99 ± 9%; was respectively obtained from river, tap and mineral water, suggesting that the sensor can be used under real sample conditions. The assay was also shown to work for sensing the ochratoxin A and d-arginine vasopressin compounds, revealing its simplicity and generalizability potentialities.
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Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Mercúrio , Nanopartículas Metálicas , Colorimetria/métodos , Nanopartículas Metálicas/química , Técnicas Biossensoriais/métodos , Ouro/química , Cloreto de Sódio , DNA/química , Peptídeos , Cloreto de Sódio na Dieta , Aptâmeros de Nucleotídeos/químicaRESUMO
BACKGROUND: Studies characterizing associations between phenols, phthalates and thyroid hormones during pregnancy produce inconsistent results. This divergence may be partly attributable to false positives due to multiple comparison testing of large numbers of chemicals, and measurement error as studies rely on small numbers of biospecimens despite high intra-individual variability in urinary chemical metabolite concentrations. OBJECTIVES: This study employs a priori chemical filtering and expanded urinary biomonitoring to evaluate associations between phenol/phthalate exposures and serum thyroid hormones assessed during pregnancy. METHODS: A two-tiered approach was implemented: a) In vitro high-throughput screening results from the ToxCast/Tox21 database, as informed by a thyroid Adverse Outcome Pathway network, were evaluated to select phenols/phthalates with activity on known and putative molecular initiating events in the thyroid pathway; and b) Adjusted linear regressions were used to study associations between filtered compounds and serum thyroid hormones measured in 437 pregnant women recruited in Grenoble area (France) between 2014 and 2017. Phenol/phthalate metabolites were measured in repeated spot urine sample pools (median: 21 samples/women). RESULTS: The ToxCast/Tox21 screening reduced the chemical set from 16 to 13 and the associated number of statistical comparisons by 19%. Parabens were negatively associated with free triiodothyronine (T3) and the T3/T4 (total thyroxine) ratio. Monobenzyl phthalate was positively associated with total T4 and negatively with the T3/T4 ratio. Effect modification by iodine status was detected for several compounds (among them ΣDEHP and mono-n-butyl phthalate) that were associated with some hormones among women with normal iodine levels. CONCLUSION: For these chemicals, screening for compounds with an increased likelihood for thyroid-related effects and relying on repeated urine samples to assess exposures improved the overall performance of multichemical analyses of thyroid disruption. This approach may improve future evaluations of human data for the thyroid pathway with implication for fetal health and may serve as a model for evaluating other toxicity outcomes. https://doi.org/10.1289/EHP10239.
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Rotas de Resultados Adversos , Iodo , Ácidos Ftálicos , Feminino , Humanos , Gravidez , Glândula Tireoide , Fenol , Ácidos Ftálicos/urina , Hormônios Tireóideos , Fenóis/urinaRESUMO
We describe herein an aptamer-based sensing approach that signal the presence of small-molecule targets when fluorescent DNA probes are challenged with the Ni2+ or Co2+ quencher metal ions. Functional oligonucleotides targeting L-tyrosinamide (L-Tym), adenosine (Ade) or cocaine (Coc) were end-labeled by the Texas-Red fluorophore. A fluorescence quenching occurred upon association of these transition metal ions with the free conjugates. The formation of the target-probe complex, by the way of variations in the overall binding of quencher metal ions along the DNA strands, led to a partial restoration (for the Ade and Coc systems) or a further attenuation (for the L-Tym system) of the fluorescence intensity. The absolute signal gain varied from 40 to 180% depending on the target-probe pair investigated. The approach was also used to detect the compound Ade in a spiked biological matrix in 1 min or less. The transition metal ion-based quenching strategy is characterized by its very simple implementation, low cost, and rapid signaling.
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Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Aptâmeros de Nucleotídeos/química , Polarização de Fluorescência , Corantes Fluorescentes/química , ÍonsRESUMO
Glioblastoma (GBM) is the most aggressive malignant glioma, with a very poor prognosis; as such, efforts to explore new treatments and GBM's etiology are a priority. We previously described human GBM cells (R2J-GS) as exhibiting the properties of cancer stem cells (growing in serum-free medium and proliferating into nude mice when orthotopically grafted). Sodium selenite (SS)-an in vitro attractive agent for cancer therapy against GBM-was evaluated in R2J-GS cells. To go further, we launched a preclinical study: SS was given orally, in an escalation-dose study (2.25 to 10.125 mg/kg/day, 5 days on, 2 days off, and 5 days on), to evaluate (1) the absorption of selenium in plasma and organs (brain, kidney, liver, and lung) and (2) the SS toxicity. A 6.75 mg/kg SS dose was chosen to perform a tumor regression assay, followed by MRI, in R2J-GS cells orthotopically implanted in nude mice, as this dose was nontoxic and increased brain selenium concentration. A group receiving TMZ (5 mg/kg) was led in parallel. Although not reaching statistical significance, the group of mice treated with SS showed a slower tumor growth vs. the control group (p = 0.08). No difference was observed between the TMZ and control groups. We provide new insights of the mechanisms of SS and its possible use in chemotherapy.
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Neoplasias Encefálicas/tratamento farmacológico , Corpo Estriado/cirurgia , Glioblastoma/tratamento farmacológico , Células-Tronco Neoplásicas/transplante , Selenito de Sódio/efeitos adversos , Oligoelementos/efeitos adversos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Corpo Estriado/metabolismo , Glioblastoma/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Selênio/metabolismo , Selenito de Sódio/administração & dosagem , Temozolomida/administração & dosagem , Oligoelementos/administração & dosagem , Resultado do TratamentoRESUMO
We introduced an aptamer switch design that relies on the ability of post-transition/transition metal ions to trigger, through their coordination to nucleobases, substantial DNA destabilization. In the absence of molecular target, the addition of one such metal ion to usual aptamer working solutions promotes the formation of an alternative, inert DNA state. Upon exposure to the cognate compound, the equilibrium is shifted towards the competent DNA form. The switching process was preferentially activated by metal ions of intermediate base over phosphate complexation preference (i.e. Pb2+ , Cd2+ ) and operated with diversely structured DNA molecules. This very simple aptamer switch scheme was applied to the detection of small organics using the fluorescence anisotropy readout mode. We envision that the approach could be adapted to a variety of signalling methods that report on changes in the surface charge density of DNA receptors.
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Many similarities between tryptophan (Trp) and phenylalanine (Phe) metabolisms exist. It is possible that a modification of Trp metabolism might be seen in phenylketonuria (PKU). As some of these metabolites have neuroactive properties, they should be consider in neurological impairment seen in this pathology and not totally explained by blood Phe concentrations. One hundred and fifty-one adult PKU patients (mean age 26.8 years) were included for this study. Plasma Trp, kynurenine (KYN), 3-hydroxykynurenic acid (3HK), and kynurenic acid (KA) were analyzed by liquid chromatography coupled with tandem mass spectrometry. KYN and 3HK were significantly lower in PKU patients compared to general population (P < .0001), and KA was significantly enhanced is this population (P = .009). Furthermore, 3HK concentration was significantly different between PKU patients underwent controlled low-Phe diet compared to PKU patients without this diet (P = .0016). In PKU patients with diet, taking AA substitute enable higher plasma 3HK concentration than without (P = .0008) but still not reaching general population level (P < .0001). Although further study has to be done, it is clear that Trp metabolism is modified in adult PKU patients. An exploration of complete Trp metabolism, and not only Trp concentration, is needed in PKU population, but also in other inborn error of metabolism treated with hypoprotidic diet.
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Fenilcetonúrias/sangue , Triptofano/metabolismo , Adulto , Análise Química do Sangue/métodos , Cromatografia Líquida , Feminino , França , Humanos , Masculino , Fenilcetonúrias/diagnóstico , Fenilcetonúrias/metabolismo , Estudos Prospectivos , Espectrometria de Massas em TandemRESUMO
The development of enantioselective assays and sensors has received much attention for the determination of enantiomeric impurities. Herein, we demonstrated that the previously reported aptamer kissing complex (AKC) assay strategy can be implemented for designing a chiral tool that allows both the simultaneous enantiomer quantification and the enantiopurity analysis. D- and L-arginine vasopressin (AVP) were employed as model enantiomeric targets. The D- and L-AVP engineered aptamers (aptaswitch) were used as recognition units whereas the Fluorescein or Texas Red labelled D- and L-hairpin probes (aptakiss) served as probes of the enantiomer-dependent AKC formation. The orthogonal fluorescence anisotropy signaling scheme at two different emission wavelengths permitted the concomitant sensing of the AVP enantiomers in a single sample, under a high-throughput microplate format. It was also shown that the AKC-based enantioselective sensor allowed the enantiomeric impurity detection at a level as low as 0.01%.
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The aim of this study was to assess the validity of the predictive INTERSALT equation using spot urine samples to estimate 24-h urinary Na (24-hUNa) excretion and daily Na intake among the French adult population. Among 193 French adults ('validation sample'), we assessed the validity by comparing predicted 24-hUNa excretion from spot urine and measured 24-hUNa excretion from 24-h urine collections. Spearman correlation coefficients and Bland-Altman plots were used and we calculated calibration coefficients. In a nationally representative sample of 1720 French adults ('application sample'), the calibrated predictive equation was then applied to the spot urine Na values to estimate 24-hUNa excretion and daily Na intake. In that sample, predicted Na intake was compared with that estimated from 24-h dietary recalls. Results were adjusted and corrected using calibration coefficients. In the validation sample, the measured 24-hUNa excretion was on average 14 % higher than the predicted 24-hUNa (+13 % for men and +16 % for women). Correlation between measured and predicted 24-hUNa excretion was moderate (Spearman r 0·42), and the Bland-Altman plots showed underestimation at lower excretion level and overestimation at higher level. In the application study, estimated daily salt intake was 8·0 g/d using dietary recalls, 8·1 g/d using predicted INTERSALT equation and 9·3 g/d after applying calibration coefficients calculated in the validation study. Despite overall underestimation of 24-hUNa excretion by spot urinary Na, the use of predictive INTERSALT equation remains an acceptable alternative in monitoring global Na intake/excreted in the French population but its use is not advised at the individual level.
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Sódio na Dieta/administração & dosagem , Sódio/urina , Adulto , Idoso , Dieta , Registros de Dieta , Reações Falso-Negativas , Feminino , França , Humanos , Masculino , Rememoração Mental , Pessoa de Meia-Idade , Inquéritos Nutricionais , Fatores de Tempo , Coleta de Urina/métodosAssuntos
Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/genética , Oxirredução , Adulto , Idoso , Antimicina A/farmacologia , Antioxidantes/metabolismo , Feminino , Humanos , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/mortalidade , Peroxidação de Lipídeos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , Oniocompostos/farmacologia , Prognóstico , Modelos de Riscos Proporcionais , Estudos Prospectivos , Espécies Reativas de Oxigênio/metabolismo , Rotenona/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Translocação GenéticaRESUMO
Glioblastoma multiform (GBM) tumors are very heterogeneous, organized in a hierarchical pattern, including cancer stem cells (CSC), and are responsible for development, maintenance, and cancer relapse. Therefore, it is relevant to establish new GBM cell lines with CSC characteristics to develop new treatments. A new human GBM cell line, named R2J, was established from the cerebro-spinal fluid (CSF) of a patient affected by GBM with leptomeningeal metastasis. R2J cells exhibits an abnormal karyotype and form self-renewable spheres in a serum-free medium. Original tumor, R2J, cultured in monolayer (2D) and in spheres showed a persistence expression of CD44, CD56 (except in monolayer), EGFR, Ki67, Nestin, and vimentin. The R2J cell line is tumorigenic and possesses CSC properties. We tested in vitro the anticancer effects of sodium selenite (SS) compared to temozolomide TMZ. SS was absorbed by R2J cells, was cytotoxic, induced an oxidative stress, and arrested cell growth in G2M before inducing both necrosis and apoptosis via caspase-3. SS also modified dimethyl-histone-3-lysine-9 (H3K9m2) levels and decreased histone deacetylase (HDAC) activity, suggesting anti-invasiveness potential. This study highlights the value of this new GBM cell line for preclinical modeling of clinically relevant, patient specific GBM and opens a therapeutic window to test SS to target resistant and recurrent GBM.
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OBJECTIVE: Most patients with resistant hypertension (RH) have obstructive sleep apnea (OSA). We aimed to determine the impact of OSA and continuous positive airway pressure (CPAP) treatment on the leptin profile and blood pressure (BP) in patients with RH. METHODS: After an initial case-control study (RH with and without OSA), we performed a randomized, single blind study in OSA + RH patients receiving either sham CPAP (3 months) followed by active CPAP (6 months) or 6 months of active CPAP. The primary outcome was the comparison of leptin levels between groups of RH patients with or without OSA. Secondary outcomes were the comparison of metabolic parameters, biomarkers of sympathetic activity, and BP indices between the two groups of RH patients with or without OSA. The same outcomes were then evaluated and compared before and after sham and effective CPAP intervention. RESULTS: Sixty-two patients (60 ± 10 years; 77% men) with RH (24-h daytime systolic BP (SBP)/diastolic BP: 145 ± 13/85 ± 10 mmHg, 3.7 antihypertensive drugs) were included. The 37 RH patients exhibiting OSA (60%) were predominantly men (87 vs 64% for non-OSA patients), with a greater prevalence of metabolic syndrome and higher creatininemia. Their leptin concentrations were significantly lower than in non-OSA patients [9 (6; 15) vs 17 (6; 29) ng/mL] but increased after 6 months of CPAP. Three months of effective CPAP significantly decreased night-time SBP by 6.4 mmHg and heart rate (HR) by 6.0 bpm, compared to sham CPAP. CONCLUSION: The association between OSA and RH corresponds to a specific, predominately male phenotype with a higher burden of metabolic syndrome and higher creatininemia but there was no significant difference between OSA and non-OSA patients regarding BP indices, and the number of antihypertensive drugs used. Active CPAP could be efficient at decreasing night-time BP and HR, but there was no difference between CPAP and sham CPAP groups for all metabolic and SNS markers (NCT00746902 RHOOSAS).
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Phenylketonuria (PKU, OMIM 261600) is an autosomal recessive inborn error of metabolism caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH; EC 1.14.16.1). If untreated, the disease leads to an important intellectual disability (IQ <50). Although many facts are common between phenylalanine (Phe) and tryptophan (Trp) metabolism, little is known about Trp metabolism modification in PKU. Our aim was to evaluate the modifications of Trp metabolism in a phenylketonuric population. A monocentric study was conducted between October 2016 and March 2017. Every phenylketonuric fasting adults were included during their annual follow up. For each patient, 9 analytes of Trp metabolism were quantified in peripheral blood using liquid chromatography coupled with tandem mass spectrometry. Mann and Whitney tests (p <0.05) were carried out in StatView 5.0 software. A total of 6 PKU patients were studied. Significant modification of Trp metabolism was shown. Indeed, three analytes, i.e. tryptophan, kynurenine and 3-hydroxykynurenic acid, were significantly lower in phenylketonuric than in healthy population (p-value <0.05), without known confounding factors. This study shows a significant modification of Trp metabolism in peripheral blood of phenylketonuric patients. Nevertheless, more investigations are necessary to confirm the modification of Trp metabolism in PKU and to determine how this metabolism is involved in neurological symptoms.