Noncompetitive Determination of Small Analytes by Sandwich-Type Lateral Flow Assay Based on an Aptamer Kissing Complex.

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.

A novel injectable radiopaque hydrogel with potent properties for multicolor CT imaging in the context of brain and cartilage regenerative therapy.

Cell therapy is promising to treat many conditions, including neurological and osteoarticular diseases. Encapsulation of cells within hydrogels facilitates cell delivery and can improve therapeutic effects. However, much work remains to be done to align treatment strategies with specific diseases. The development of imaging tools that enable monitoring cells and hydrogel independently is key to achieving this goal. Our objective herein is to longitudinally study an iodine-labeled hydrogel, incorporating gold-labeled stem cells, by bicolor CT imaging after in vivo injection in rodent brains or knees. To this aim, an injectable self-healing hyaluronic acid (HA) hydrogel with long-persistent radiopacity was formed by the covalent grafting of a clinical contrast agent on HA. The labeling conditions were tuned to achieve sufficient X-ray signal and to maintain the mechanical and self-healing properties as well as injectability of the original HA scaffold. The efficient delivery of both cells and hydrogel at the targeted sites was demonstrated by synchrotron K-edge subtraction-CT. The iodine labeling enabled to monitor the hydrogel biodistribution in vivo up to 3 days post-administration, which represents a technological first in the field of molecular CT imaging agents. This tool may foster the translation of combined cell-hydrogel therapies into the clinics.

Exposure to a mixture of non-persistent environmental chemicals and neonatal thyroid function in a cohort with improved exposure assessment.

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.

Nile blue as reporter dye in salt aggregation based-colorimetric aptasensors for peptide, small molecule and metal ion detection.

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.

Intracellular accumulation and immunological response of NIR-II polymeric nanoparticles.

Polymeric nanoparticles (NPs) are extremely promising for theranostic applications. However, their interest depends largely on their interactions with immune system, including the capacity to activate inflammation after their capture by macrophages. In the present study, we generated monodisperse poly(ethyl methacrylate) (PEMA) NPs loaded with hydrophobic photoluminescent gold nanoclusters (Au NCs) emitting in the NIR-II optical windows and studied their interaction in vitro with J774.1A macrophages. PEMA NPs showed an efficient time and dose dependent cellular uptake with up to 70 % of macrophages labelled in 24 h without detectable cell death. Interestingly, PEMA and Au-PEMA NPs induced an anti-inflammatory response and a strong down-regulation of nitric oxide level on lipopolysacharides (LPS) activated macrophages, but without influence on the levels of reactive oxygen species (ROS). These polymeric NPs may thus present a potential interest for the treatment of inflammatory diseases.

Hypodopaminergic state of the nigrostriatal pathway drives compulsive alcohol use.

The neurobiological mechanisms underlying compulsive alcohol use, a cardinal feature of alcohol use disorder, remain elusive. The key modulator of motivational processes, dopamine (DA), is suspected to play an important role in this pathology, but its exact role remains to be determined. Here, we found that rats expressing compulsive-like alcohol use, operationalized as punishment-resistant self-administration, showed a decrease in DA levels restricted to the dorsolateral territories of the striatum, the main output structure of the nigrostriatal DA pathway. We then causally demonstrated that chemogenetic-induced selective hypodopaminergia of this pathway resulted in compulsive-like alcohol self-administration in otherwise resilient rats, accompanied by the emergence of alcohol withdrawal-like motivational impairments (i.e., impaired motivation for a natural reinforcer). Finally, the use of the monoamine stabilizer OSU6162, previously reported to correct hypodopaminergic states, transiently decreased compulsive-like alcohol self-administration in vulnerable rats. These results suggest a potential critical role of tonic nigrostriatal hypodopaminergic states in alcohol addiction and provide new insights into our understanding of the neurobiological mechanisms underlying compulsive alcohol use.

ATP7B-Deficient Hepatocytes Reveal the Importance of Protein Misfolding Induced at Low Copper Concentration.

Copper is a transition metal essential for human life. Its homeostasis is regulated in the liver, which delivers copper to the whole body and excretes its excess outside the organism in the feces through the bile. These functions are regulated within hepatocytes, and the ATP7B copper transporter is central to making the switch between copper use and excretion. In Wilson disease, the gene coding for ATP7B is mutated, leading to copper overload, firstly, in the liver and the brain. To better understand the role of ATP7B in hepatocytes and to provide a smart tool for the development of novel therapies against Wilson disease, we used the CrispR/Cas9 tool to generate hepatocyte cell lines with the abolished expression of ATP7B. These cell lines revealed that ATP7B plays a major role at low copper concentrations starting in the micromolar range. Moreover, metal stress markers are induced at lower copper concentrations compared to parental cells, while redox stress remains not activated. As shown recently, the main drawback induced by copper exposure is protein unfolding that is drastically exacerbated in ATP7B-deficient cells. Our data enabled us to propose that the zinc finger domain of DNAJ-A1 would serve as a sensor of Cu stress. Therefore, these Wilson-like hepatocytes are of high interest to explore in more detail the role of ATP7B.

Phenol and Phthalate Effects on Thyroid Hormone Levels during Pregnancy: Relying on In Vitro Assays and Adverse Outcome Pathways to Inform an Epidemiological Analysis.

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.

Detection of small molecules by fluorescence intensity using single dye labeled aptamers and quencher transition metal ions.

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.

Preclinical Evaluation of Sodium Selenite in Mice: Toxicological and Tumor Regression Studies after Striatum Implantation of Human Glioblastoma Stem Cells.

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.

Theranostic AGuIX nanoparticles as radiosensitizer: A phase I, dose-escalation study in patients with multiple brain metastases (NANO-RAD trial).

BACKGROUND AND PURPOSE: Brain metastasis impacts greatly on patients' quality of life and survival. The phase I NANO-RAD trial assessed the safety and maximum tolerated dose of systemic administration of a novel gadolinium-based nanoparticle, AGuIX, in combination with whole brain radiotherapy in patients with multiple brain metastases not suitable for stereotactic radiotherapy. MATERIALS AND METHODS: Patients with measurable brain metastases received escalating doses of AGuIX nanoparticles (15, 30, 50, 75, or 100 mg/kg intravenously) on the day of initiation of WBRT (30 Gy in 10 fractions) in 5 cohorts of 3 patients each. Toxicity was assessed using NCI Common Terminology Criteria for Adverse Events v4.03. RESULTS: Fifteen patients with 354 metastases were included. No dose-limiting toxic effects were observed up to AGuIX 100 mg/kg. Plasma elimination half-life of AGuIX was similar for all groups (mean 1.3 h; range 0.8-3 h). Efficient targeting of metastases (T1 MRI enhancement, tumor selectivity) and persistence of AGuIX contrast enhancement were observed in metastases from patients with primary melanoma, lung, breast, and colon cancers. The concentration of AGuIX in metastases after administration was proportional to the injected dose. Thirteen of 14 evaluable patients had a clinical benefit, with either stabilization or reduction of tumor volume. MRI analysis showed significant correlation between contrast enhancement and tumor response, thus supporting a radiosensitizing effect. CONCLUSION: Combining AGuIX with radiotherapy for patients with brain metastases is safe and feasible. AGuIX specifically targets brain metastases and is retained within tumors for up to 1 week; ongoing phase II studies will more definitively assess efficacy.

Anti-pesticide DNA aptamers fail to recognize their targets with asserted micromolar dissociation constants.

Herein, we originally aimed at developing fluorescence anisotropy biosensor platforms devoted to the homogeneous-phase detection of isocarbophos and phorate pesticides by using previously isolated DNA aptamers. To achieve this, two reporting approaches displaying very high generalizability features were implemented, based on either the complementary strand or the SYBR green intercalator displacement strategies. Unfortunately, none of the transduction methods led to phorate-dependent signals. Only the SYBR green displacement method provided a small output in the presence of isocarbophos, but at an analyte concentration greater than 100 µM. In order to identify the origin of such data, isothermal titration calorimetry (ITC) experiments were subsequently performed. It was shown that aptamers bind neither isocarbophos nor phorate in free solution with the claimed micromolar dissociation constants. This work puts forward some doubts about the previously described aptasensors that rely on the use of these functional DNA molecules. It also highlights the need to carefully investigate the binding capabilities of aptamers after their isolation and to include appropriate control experiments with scrambled or mutated oligonucleotides.

Aptamer Switches Regulated by Post-Transition/Transition Metal Ions.

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.

Surface functionalization of gold nanoclusters with arginine: a trade-off between microtumor uptake and radiotherapy enhancement.

Ultra-small gold nanoclusters (AuNCs) are increasingly investigated for cancer imaging and radiotherapy enhancement. While fine-tuning the AuNC surface chemistry can optimize their pharmacokinetics, its effects on radiotherapy enhancement remain largely unexplored. This study demonstrates that optimizing the surface chemistry of AuNCs for increased tumor uptake can significantly affect its potential to augment radiotherapy outcomes.

High-Resolution Shortwave Infrared Imaging of Vascular Disorders Using Gold Nanoclusters.

We synthesized a generation of water-soluble, atomically precise gold nanoclusters (Au NCs) with anisotropic surface containing a short dithiol pegylated chain (AuMHA/TDT). The AuMHA/TDT exhibit a high brightness (QY ∼ 6%) in the shortwave infrared (SWIR) spectrum with a detection above 1250 nm. Furthermore, they show an extended half-life in blood (t1/2ß = 19.54 ± 0.05 h) and a very weak accumulation in organs. We also developed a non-invasive, whole-body vascular imaging system in the SWIR window with high-resolution, benefiting from a series of Monte Carlo image processing. The imaging process enabled to improve contrast by 1 order of magnitude and enhance the spatial resolution by 59%. After systemic administration of these nanoprobes in mice, we can quantify vessel complexity in depth (>4 mm), allowing to detect very subtle vascular disorders non-invasively in bone morphogenetic protein 9 (Bmp9)-deficient mice. The combination of these anisotropic surface charged Au NCs plus an improved SWIR imaging device allows a precise mapping at high-resolution and an in depth understanding of the organization of the vascular network in live animals.

Kissing interactions for the design of a multicolour fluorescence anisotropy chiral aptasensor.

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%.

Investigating the toxic effects induced by iron oxide nanoparticles on neuroblastoma cell line: an integrative study combining cytotoxic, genotoxic and proteomic tools.

Nanomaterials have gained much attention for their use and benefit in several fields. Iron Oxide Nanoparticles (IONPs) have been used in Biomedicine as contrast agents for imaging cancer cells. However, several studies reported the potential toxicity of those nanoparticles in different models, especially in cells. Therefore, in our present study, we investigated the effects of IONPs on the SH-SY5Y neuroblastoma cell line. We carried out cytotoxic and genotoxic studies to evaluate the phenotypic effects, and proteomic investigation to evaluate the molecular effects and the mechanisms by which this kind of NPs could induce toxicity. Our results showed that the use of three different sizes of IONPs (14, 22 and 30 nm) induced cell detachment, cell morphological changes, size, and concentration-dependent IONP internalization and cell mortality. IONPs induced slight genotoxic damage assayed by modified comet assay without affecting cell cycle, mitochondrial function, membrane integrity, intracellular calcium level, and without inducing ROS generation. All the studies were performed to compare also the effects of IONPs to the ferric iron by incubating cells with equivalent concentration of FeCl3. In all tests, the NPs exhibited more toxicity than the ferric iron. The proteomic analysis followed by gene ontology and pathway analysis evidenced the effects of IONPs on cytoskeleton, cell apoptosis, and cancer development. Our findings provided more information about IONP effects on human cells and especially on cancer cell line.

Nanoparticles in foods? A multiscale physiopathological investigation of iron oxide nanoparticle effects on rats after an acute oral exposure: Trace element biodistribution and cognitive capacities.

Iron Oxide Nanoparticles (IONPs) are used in several fields of application, mainly in the biomedical field for their magnetic properties and in food additive known as "E172" for their colour. In the present investigation, we focused on IONP effects on Wistar rat following acute oral exposure. We performed a multiscale physiopathological investigation in order to elucidate potential toxic effects linked to IONP ingestion, especially on cognitive capacities, trace element distribution, blood constituents, organ functions, organ structure and iron deposit. We demonstrated that oral exposure to IONPs induces disturbances of certain parameters depending on the dose. Interestingly, the histopathological examination evidenced inflammatory effects of IONPs in the liver with iron deposits in hepatocytes and Kuppfer cells. Neurobehavioral examination showed that oral exposure to IONPs did not affect nor rat emotions, exploration and locomotion capacities, nor spatial reference memory status. Furthermore, oral administration of IONPs did not disrupt the trace element homeostasis nor in the liver neither in the stomach. Altogether, our study evidenced low signs of toxicity, but some effects lead us to a careful use of these NPs. Thereby, their use in foods should be further studied to better evaluate the potential toxic risks of the oral exposure to IONPs.