An aqueous one-pot route to gold/quantum rod heterostructured nanoparticles functionalized with DNA.

We report an original approach exploiting the photoelectrochemical properties of quantum rods and the versatility of Au(I) organometallic chemistry to control DNA surface grafting. This one-pot aqueous approach provides Janus biofunctionalized nanoparticles, the assembly of which should results in the emergence of synergistic properties.

Identification of two-step chemical mechanisms using small temperature oscillations and a single tagged species.

In order to identify two-step chemical mechanisms, we propose a method based on a small temperature modulation and on the analysis of the concentration oscillations of a single tagged species involved in the first step. The thermokinetic parameters of the first reaction step are first determined. Then, we build test functions that are constant only if the chemical system actually possesses some assumed two-step mechanism. Next, if the test functions plotted using experimental data are actually even, the mechanism is attributed and the obtained constant values provide the rate constants and enthalpy of reaction of the second step. The advantage of the protocol is to use the first step as a probe reaction to reveal the dynamics of the second step, which can hence be relieved of any tagging. The protocol is anticipated to apply to many mechanisms of biological relevance. As far as ligand binding is considered, our approach can address receptor conformational changes or dimerization as well as competition with or modulation by a second partner. The method can also be used to screen libraries of untagged compounds, relying on a tracer whose concentration can be spectroscopically monitored.

Identification of two-step chemical mechanisms and determination of thermokinetic parameters using frequency responses to small temperature oscillations.

Increased focus on kinetic signatures in biology, coupled with the lack of simple tools for chemical dynamics characterization, lead us to develop an efficient method for mechanism identification. A small thermal modulation is used to reveal chemical dynamics, which makes the technique compatible with in cellulo imaging. Then, the detection of concentration oscillations in an appropriate frequency range followed by a judicious analytical treatment of the data is sufficient to determine the number of chemical characteristic times, the reaction mechanism, and the full set of associated rate constants and enthalpies of reaction. To illustrate the scope of the method, dimeric protein folding is chosen as a biologically relevant example of nonlinear mechanism with one or two characteristic times.

Chemical mechanism identification from frequency response to small temperature modulation.

The description of interactions between biochemical species and the elucidation of the corresponding chemical mechanisms encounter an increasing interest both for the comprehension of biological pathways at the molecular scale and for the rationalization of drug design. Relying on powerful experimental tools such as thermal microfluidics and fluorescence detection, we propose a methodology to determine the chemical mechanism of a reaction without fitting parameters. A mechanism consistent with the accessible knowledge is assumed, and the assumption is checked through an iterative protocol. The test is based on the frequency analysis of the response of a targeted reactive species to temperature modulation. We build specific functions of the frequency that are constant for the assumed mechanism and show that the graph of these functions can be drawn from appropriate data analysis. The method is general and can be applied to any complex mechanism. It is here illustrated in detail in the case of single relaxation time mechanisms.

Temperature modulation and quadrature detection for selective titration of two-state exchanging reactants.

Biological samples exhibit huge molecular diversity over large concentration ranges. Titrating a given compound in such mixtures is often difficult, and innovative strategies emphasizing selectivity are thus demanded. To overcome limitations inherent to thermodynamics, we here present a generic technique where discrimination relies on the dynamics of interaction between the target of interest and a probe introduced in excess. Considering an ensemble of two-state exchanging reactants submitted to temperature modulation, we first demonstrate that the amplitude of the out-of-phase concentration oscillations is maximum for every compound involved in a reaction whose equilibrium constant is equal to unity and whose relaxation time is equal to the inverse of the excitation angular frequency. Taking advantage of this feature, we next devise a highly specific detection protocol and validate it using a microfabricated resistive heater and an epifluorescence microscope, as well as labeled oligonucleotides to model species displaying various dynamic properties. As expected, quantification of a sought for strand is obtained even if interfering reagents are present in similar amounts. Moreover, our approach does not require any separation and is compatible with imaging. It could then benefit some of the numerous binding assays performed every day in life sciences.

Fluorescent thermometers for dual-emission-wavelength measurements: molecular engineering and application to thermal imaging in a microsystem.

To facilitate thermal imaging, particularly in microdevices, one has to favor molecular thermometers in which the response is independent of the probe concentration and of the observation setup imperfections. Hence, this paper introduces two temperature fluorescent probes for ratiometric dual-emission-wavelength measurements in aqueous solutions. They are based on a nonathermal chemical reaction, either a conformational transition or a protonation, that induces a modification of their emission spectra as the temperature changes. Relying on both a straightforward theoretical analysis and thorough photophysical, thermodynamic, and kinetic investigations, we demonstrate how the flexible design of these two thermometers can be optimized to face applications with various requirements in terms of operating temperature and wavelength ranges as well as temporal resolution. For instance, the present molecules, which can be used between 5 and 35 degrees C, provide a relative sensitivity up to approximately 9 x 10(-2) K(-1) and milli- to microsecond response times. Finally, we utilize a two-color molecular beacon, a probe belonging to the first series of thermometers, to image temperature profiles in a microfluidic cell heated by a resistive strip. The ratiometric analysis of the fluorescence emission at two different wavelengths is performed on a widely available dual-view microscope, illustrating both the simplicity and reliability of the thermal mapping protocol.

Fourier analysis to measure diffusion coefficients and resolve mixtures on a continuous electrophoresis chip.

We report a method to measure diffusion coefficients of fluorescent solutes in the 10(2)-10(6) Da molecular mass range in a glass-PDMS chip. Upon applying a permanent electric field, the solute is introduced through a narrow channel into a wide analysis chamber where it migrates along the injection axis and diffuses in two dimensions. The diffusion coefficient is extracted after 1D Fourier transform of the resulting stationary concentration pattern. Analysis is straightforward, requiring no numerical integration or velocity field simulation. The diffusion coefficients measured for fluorescein, rhodamine green-labeled oligonucleotides, and YOYO-1-stained dsDNA fragments agree with the literature values and with our own fluorescence correlation spectroscopy measurements. As shown for 151 and 1257 base pair dsDNA mixtures, the present method allows us to rely on diffusion to quantitatively characterize the nature and the composition of binary mixtures. In particular, we implement a DNA hybridization assay to illustrate the efficiency of the proposed protocol for library screening.

Quadruplex-based molecular beacons as tunable DNA probes.

Molecular beacons (MBs) are fluorescent nucleic acid probes with a hairpin-shaped structure in which the 5' and 3' ends are self-complementary. Due to a change in their emissive properties upon recognition with complementary sequences, MBs allow the diagnosis of single-stranded DNA or RNA with high mismatch discrimination, in vitro and in vivo. Whereas the stems of MB hairpins usually rely on the formation of a Watson-Crick duplex, we demonstrate in this report that the preceding structure can be replaced by a G-quadruplex motif (G4). Intramolecular quadruplexes may still be formed with a central loop composed of 12 to 21 bases, therefore extending the sequence repertoire of quadruplex formation. G4-MB can efficiently be used for oligonucleotide discrimination: in the presence of a complementary sequence, the central loop hybridizes and forms a duplex that causes opening of the quadruplex stem. The corresponding G4-MB unfolding can be detected by a change in its fluorescence emission. We discuss the thermodynamic and kinetic opportunities that are provided by using G4-MB instead of traditional MB. In particular, the intrinsic feature of the quadruplex motif facilitates the design of functional molecular beacons by independently varying the concentration of monovalent or divalent cations in the medium.

Magnetic force probe for nanoscale biomolecules.

We present a new technique to measure the mechanical properties of small biomolecules. This technique uses long range repulsive colloidal forces together with magnetic attraction as a force probing tool. The biomolecules are grafted between superparamagnetic particles, which are regularly spaced within long chains maintained by an external magnetic field. Varying the magnetic field results in compression or extension of the molecules between the particles. In order to demonstrate this technique we use, as a size controlled model molecule, a short double stranded DNA (151 base pairs) for which the force-extension law is determined and found in agreement with existing predictions.

Micelles of lipid-oligonucleotide conjugates: implications for membrane anchoring and base pairing.

This report examines the organization properties of new fluorescent DNA-lipids, either alone in water or in interaction with 1-octyl-beta-d-glucopyranoside micelles or egg phosphatidylcholine vesicles. We first describe the design and the syntheses of the conjugates. Then, we use UV-Vis absorption, steady-state fluorescence emission, electron microscopy, and fluorescence correlation spectroscopy after two-photon excitation to show that these DNA-lipids form spherical micelles in aqueous solution and incorporate much better in micelles than in vesicles. We also investigate the significance of the lipophilic chains of these DNA-lipids on the melting behavior of the double-stranded hybrids: in water melting curves are broadened whereas in amphiphilic assemblies duplexes melt as the unconjugated controls. This work is expected to be useful for improving the rational design of antisense medicines.

Selective adhesion, lipid exchange and membrane-fusion processes between vesicles of various sizes bearing complementary molecular recognition groups.

Equimolar mixtures of large unilamellar vesicles (LUVs) obtained from mixtures of egg lecithin and lipids containing complementary hydrogen bonding head groups (barbituric acid (BAR) and 2,4,6-triaminopyrimidine (TAP)) were shown to aggregate and fuse. These events have been studied in detail using electron microscopy and dynamic light scattering, and by fluorimetry using membrane or water-soluble fluorescence probes. It was shown that aggregation was followed by two competitive processes: a) lipid mixing leading to redispersion of the vesicles; b) fusion events generating much larger vesicles. In order to better understand the nature of the interaction, the effects of ionic strength and surface concentration of recognition lipids on the aggregation process were investigated by dynamic light scattering. Additionally, it was possible to inhibit the aggregation kinetics through addition of a soluble barbituric acid competitor. The study was extended to giant unilamellar vesicles (GUVs) to investigate the size effect and visualise the phenomena in situ. The interactions between complementary LUVs and GUVs or GUVs and GUVs were studied by optical microscopy using dual fluorescent labelling of both vesicle populations. A selective adhesion of LUVs onto GUVs was observed by electron and optical microscopies, whereas no aggregation took place in case of a GUV/GUV mixture. Furthermore, a fusion assay of GUV and LUV using the difference of size between GUV and LUV and calceine self-quenching showed that no mixing between the aqueous pools occured.

Catalytic action of fuculose 1-phosphate aldolase (class II) as derived from structure-directed mutagenesis.

Previous analyses established the structures of unligated L-fuculose 1-phosphate aldolase and of the enzyme ligated with an inhibitor mimicking the substrate dihydroxyacetone phosphate. These data allowed us to suggest a catalytic mechanism. On the basis of this proposal, numerous mutations were now introduced at the active center and tested with respect to their catalytic rates and their product distributions. For several mutants, the structures were determined. The results demonstrate the catalytic importance of some particular residues in defined conformations and in the mobile C-terminal chain end. Moreover, they led to a modification of the proposed mechanism. The effect of some mutations on enantioselectivity and on the ratio of diastereomer formation indicates clearly the binding site of the aldehyde moiety in relation to the other substrate dihydroxyacetone phosphate.

Chemical stability of ecomustine, a new antitumor agent in aqueous and biological media as assessed by high-performance liquid chromatography.

Ecomustine, or CY233 (NSC-609224), is a new water-soluble nitrosoureido sugar derived from acosamine. A high-performance liquid chromatographic assay (HPLC) developed to quantify the unchanged drug in aqueous solutions and biological specimens enabled us to study the chemical stability as a function of pH, light, and temperature. In buffered aqueous solutions, the kinetics of degradation of CY233 is a first-order process. The log k-pH profile demonstrated hydroxide ion-catalyzed solvolysis. The drug is most stable at pH 4, more stable than some other nitrosoureas in 5% glucose (t1/2, 62-67 h) and in 0.9% isotonic saline (t1/2, 25-37 h) solutions. Based on these findings, blood samples should be collected in cold tubes (4 degrees C) containing citrate buffer (pH 4) and all manipulations should be protected from heat and light.

Intra-arterial hepatic chemotherapy with pirarubicin. Preclinical and clinical studies.

Intra-arterial hepatic chemotherapy (IAHC) with adriamycin (ADM) has not increased its therapeutic index. For our preclinical studies, we selected pirarubicin (THP), an ADM derivative with faster cellular uptake. In rabbits with VX2 tumor in the liver we compared plasmatic and cellular pharmacokinetics of ADM and THP after i.v. and IAH therapy. For ADM, there were no differences in plasma and heart concentrations, with only a slight increase in tumoral levels after IAH compared to i.v. administration; on the other hand, with IAH THP, there was important reduction in systemic exposure with a major increase in tumoral drug distribution. In the phase I study, involving nine patients with implanted catheters, the starting dose of THP was 30 mg/m2 with a 10 mg/m2 intrapatient escalation every 3 weeks in the absence of toxicity. Pharmacokinetics were compared for i.v. and IAH administration in seven patients. The limiting toxicity was neutropenia and the maximal tolerated dose (MTD) ranged from 50 to 110 mg/m2. Moderate nausea-vomiting (grade 1-2) and alopecia (grade 1) occurred at the MTD. No arterial occlusion, gastroduodenal ulcer, hepatitis, or sclerosing cholangitis were seen. In the phase II study, in colorectal cancer patients (CRC) with metastasis confined to the liver, patients were enrolled until June 1990. THP (40 min infusion every 3 weeks) was initiated at 60 mg/m2 with 10 mg/m2 increment until grade 2 hematotoxicity. The median MTD was 85 mg/m2 (range of 60-120 mg/m2), and the median number of cycles was 7 (range of 2-11) with cumulated doses from 180 to 1,030 mg/m2. Grade 2-4 neutropenia was reached in 15 patients. Other toxicities included two arterial occlusions, one episode of gastritis, but no hepatic toxicity and no heart failure. Antitumor effect (in 18 patients) included 1 CR, 5 PR, 3 MR, 6 NC, and 3 PD. The median survival was 18+ months and 1-year survival was 73% +/- 12%. Seven patients had extrahepatic progression at this time. In conclusion, besides 5-FU or Fudr, THP is active in IAHC (probably in relation with high local extraction) on CRC liver metastases usually unresponsive to ADM. It can be given in an outpatient setting with minimal toxicity.

Characterization of the anti-tumour activity against solid tumours of a new nitrosoureido sugar: Cy 233.

The anti-tumour properties of Cy 233, a new nitrosoureido sugar, were investigated in two murine solid tumours: B16 melanoma and subcutaneously implanted colon adenocarcinoma. Injected i.v., Cy 233 exerted a strong anti-tumour effect against the established B16 melanoma: long-term survivors were recorded with all schedules of treatment. The drug was even more effective against advanced colon 38 adenocarcinoma: it produced a high percentage of total tumour regression, regardless of the route of administration (i.p., i.v., p.o.). The marked in vivo activity of Cy 233 against advanced colon 38 adenocarcinoma, which is known to be resistant to such major anti-cancer drugs as BCNU and chlorozotocin, its water solubility and its stability in aqueous media are further elements warranting toxicological and clinical studies of this agent.

Rationale for the synthesis and preliminary biological evaluation of highly active new antitumor nitrosoureido sugars.

Various new nitrosoureido derivatives of di- or trideoxy sugars were synthesized. The influence of the hydroxyl substitution pattern, the configuration at the anomeric center, and the absolute configuration of the sugar moiety on the antitumor activity of a series of nitrosoureido derivatives of di- and trideoxy sugars was studied. All compounds showed a very significant activity in vivo against L1210 leukemia, B16 melanocarcinoma, and Lewis lung carcinoma. Methyl 3-[3-(2-chloroethyl)-3-nitrosoureido]-2,3-dideoxy-alpha-D-arabino- hexopyranoside, 24 (NSC 609224), was found to be the most active compound. When treated with 24 (NSC 609224) at 20 mg/kg on day 1, at least 90% of the L1210 leukemia and B16 melanocarcinoma bearing mice showed a survival of over 60 days for a LD50 value for this compound of 42 mg/kg.

Antitumour activity of a new water-soluble nitrosoureido sugar: CY233 (NSC 609224).

L1210 leukemia was used to evaluate the antitumour activity in vivo of CY233 (NSC 609224) a new water-soluble nitrosoureido derivative of deoxysugar currently being studied in preclinical trials. The antitumour activity of CY233 is dose-dependent with the same large therapeutic index whatever the route of administration (I.P., I.V., per os). Thus starting from a single dose of 10 mg/kg (less than 25% of the LD50), 80% to 100% of mice survive at 120 days, whether the drug is being administered I.V., I.P. or P.O. These results clearly emphasize the very original and promising potentiality of CY233 among the series of alkylating agents, and more precisely nitrosoureas.

[Isolation, social support, life events and health status]. / Isolement, support social, événements de vie et état de santé.

Research on the influence of social networks and social support on health status, conducted in English-speaking countries over the past decade, is not yet widespread in France. A review of the literature reveals that this social support has undeniable effects on mental health, and less obvious effects on physical health; it also stresses the major conceptual and methodological problems encountered in socio-epidemiological approaches. It would appear necessary to construct an overall model integrating life events, coping abilities and individual psychological factors; Social support is considered as a function of social networks, fulfilling roles of emotional support, material help, information provider, egostrengthener and social normalizer. Its mechanisms for dealing with stress, especially the buffer-role hypothesis, are also discussed.