Active Acetylcholinesterase Immobilization on a Functionalized Silicon Surface.

In this work, we studied the attachment of active acetylcholinesterase (AChE) enzyme on a silicon substrate as a potential biomarker for the detection of organophosphorous (OP) pesticides. A multistep functionalization strategy was developed on a crystalline silicon surface: a carboxylic acid-terminated monolayer was grafted onto a hydrogen-terminated silicon surface by photochemical hydrosilylation, and then AChE was covalently attached through amide bonds using an activation EDC/NHS process. Each step of the modification was quantitatively characterized by ex-situ Fourier transform infrared spectroscopy in attenuated-total-reflection geometry (ATR-FTIR) and atomic force microscopy (AFM). The kinetics of enzyme immobilization was investigated using in situ real-time infrared spectroscopy. The enzymatic activity of immobilized acetylcholinesterase enzymes was determined with a colorimetric test. The surface concentration of active AChE was estimated to be Γ = 1.72 × 10(10) cm(-2).

Molecular monolayers on silicon as substrates for biosensors.

(111) silicon surfaces can be controlled down to atomic level and offer a remarkable starting point for elaborating nanostructures. Hydrogenated surfaces are obtained by oxide dissolution in hydrofluoric acid or ammonium fluoride solution. Organic species are grafted onto the hydrogenated surface by a hydrosilylation reaction, providing a robust covalent Si-C bonding. Finally, probe molecules can be anchored to the organic end group, paving the way to the elaboration of sensors. Fluorescence detection is hampered by the high refractive index of silicon. However, improved sensitivity is obtained by replacing the bulk silicon substrate by a thin layer of amorphous silicon deposited on a reflector. The development of a novel hybrid SPR interface by the deposition of an amorphous silicon-carbon alloy is also presented. Such an interface allows the subsequent linking of stable organic monolayers through Si-C bonds for a plasmonic detection. On the other hand, the semiconducting properties of silicon can be used to implement field-effect label-free detection. However, the electrostatic interaction between adsorbed species may lead to a spreading of the adsorption isotherms, which should not be overlooked in practical operating conditions of the sensor. Atomically flat silicon surfaces may allow for measuring recognition interactions with local-probe microscopy.

Semiquantitative study of the EDC/NHS activation of acid terminal groups at modified porous silicon surfaces.

Infrared spectroscopy is used to investigate the transformation of carboxyl-terminated alkyl chains immobilized on a surface into succinimidyl ester-terminated chains by reaction with an aqueous solution of N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The acid chains are covalently grafted at the surface of hydrogenated porous silicon whose large specific surface area allows for assessing the activation yield in a semiquantitative way by infrared (IR) spectroscopy and detecting trace amounts of surface products and/or reaction products of small IR cross section. In this way, we rationalize the different reaction paths and optimize the reaction conditions to obtain as pure as possible succinimidyl ester-terminated surfaces. A diagram mapping the surface composition after activation was constructed by systematically varying the solution composition. Results are accounted for by NHS surface adsorption and a kinetic competition between the various EDC-induced surface reactions.

Highly sensitive and reusable fluorescence microarrays based on hydrogenated amorphous silicon-carbon alloys.

We have designed a new architecture of fluorescent microarrays based on a thin layer of hydrogenated amorphous silicon-carbon alloy (a-Si(0.85)C(0.15):H) deposited on an aluminium-on-glass back reflector. These substrates are modified with an organic monolayer anchored through Si-C bonds and terminated with carboxyl groups, allowing for the covalent immobilization of biological probes. The fluorescence yield is maximized by optimization of the a-Si(0.85)C(0.15):H layer thickness. This approach is assessed for DNA recognition, demonstrating an increase in sensitivity by over one order of magnitude as compared to commercial slides, and the possibility of following in situ the molecular recognition event (hybridization). The immobilization chemistry provides these substrates with a superior chemical stability toward ageing or long-term exposure to physiological buffers, which allows for many successive hybridization/dehybridization cycles without measurable changes in performance.

Dose-dependent activation of distinct hypertrophic pathways by serotonin in cardiac cells.

There is substantial evidence supporting a hypertrophic action of serotonin [5-hydroxytryptamine (5-HT)] in cardiomyocytes. However, little is known about the mechanisms involved. We previously demonstrated that 5-HT-induced hypertrophy depends, in part, on the generation of reactive oxygen species by monoamine oxidase-A (MAO-A) (see Ref. 3). Cardiomyocytes express 5-HT(2) receptors, which may also participate in hypertrophy. Here, we analyzed the respective contribution of 5-HT(2) receptors and MAO-A in H9C2 cardiomyoblast hypertrophy. 5-HT induced a dose-dependent increase in [(3)H]leucine incorporation and stimulation of two markers of cardiac hypertrophy, ANF-luc and alphaSK-actin-luc reporter genes. Experiments using 1 microM 5-HT showed that hypertrophic response occurred independently from MAO-A. Using pharmacological inhibitors (M100907 and ketanserin), we identified a novel mechanism of action involving 5-HT(2A) receptors and requiring Ca(2+)/calcineurin/nuclear factor of activated T-cell activation. The activation of this hypertrophic pathway was fully prevented by 5-HT(2A) inhibitors and was unaffected by MAO inhibition. When 10 microM 5-HT was used, an additional hypertrophic response, prevented by the MAO inhibitors pargyline and RO 41-1049, was observed. Unlike the 5-HT(2A)-receptor-mediated H9C2 cell hypertrophy, MAO-A-dependent hypertrophic response required activation of extracellular-regulated kinases. In conclusion, our results show the existence of a dose-dependent shift of activation of distinct intracellular pathways involved in 5-HT-mediated hypertrophy of cardiac cells.

DNA adsorption at functionalized Si/buffer interfaces studied by x-ray reflectivity.

The adsorption of DNA on chemically homogeneous, functionalized, oxide-free single-crystal silicon surfaces is studied by x-ray reflectivity. The adsorption of monodisperse, 294 base-pair double-stranded DNA on a positively charged surface is detected through the deformation of the molecular monolayer of aminated alkyl-chain molecules covalently bonded to the surface. The adsorption of single-stranded DNA does not lead to the same deformation. A detailed quantitative characterization of the density profiles yield surface densities of the covalently grafted, molecular monolayers that are in excellent agreement with infrared spectroscopic measurements. The additional mass density that is measured following the adsorption of DNA corresponds either to the partial embedding of a densely-packed adsorbed layer or to a deeper penetration into the soft surface layer at a lower surface density of the adsorbed double-stranded DNA molecules. The adsorption is found to be irreversible under high added salt concentrations, suggesting a partial dehydration of the double-stranded DNA.

Mechanisms of thermal decomposition of organic monolayers grafted on (111) silicon.

The thermal stability of different organic layers on silicon has been investigated by in situ infrared spectroscopy, using a specially designed variable-temperature cell. The monolayers were covalently grafted onto atomically flat (111) hydrogenated silicon surfaces through the (photochemical or catalytic) hydrosilylation of 1-decene, heptadecafluoro-1-decene or undecylenic acid. In contrast to alkyl monolayers, which desorb as alkene chains around 300 degrees C by the breaking of the Si-C bond through a beta-hydride elimination mechanism, the alkyl layers functionalized with a carboxylic acid terminal group undergo successive chemical transformations. At 200-250 degrees C, the carboxyl end groups couple forming anhydrides, which subsequently decompose at 250-300 degrees C by loss of the functional group. In the case of fluorinated alkyl chains, the C-C bond located between CH2 and CF2 units is first broken at 250-300 degrees C. In either case, the remaining alkyl layer is stable up to 350 degrees C, which is accounted for by a kinetic model involving chain pairing on the surface.

Nicotine and smoker status moderate brain electric and mood activation induced by ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist.

As the increased smoking prevalence in schizophrenics may be interpreted as an adaptive response to an underlying biological defect, investigations into nicotine's actions within N-methyl-d-aspartate (NMDA) antagonist drug models of schizophrenia may improve our understanding of the role of glutamatergic neurotransmission in initiating and maintaining nicotine dependence in this disorder. In this double-blind, placebo-controlled, randomized study, the electroencephalographic (EEG) and subjective response to a sub-psychotomimetic intravenous dose of the NMDA antagonist ketamine was examined in 20 regular smokers and 20 non-smokers pretreated with placebo or nicotine gum. Although nicotine increased EEG arousal, ketamine produced electrocerebral signs of brain activation (decreased slow wave power) and sedation (decreased fast wave power and frequency), which were not affected by nicotine pretreatment and were evident only in non-smokers. Ketamine increased a number of self-report indices of subjective arousal, some of which were attenuated and potentiated by nicotine in smokers and non-smokers, respectively. These findings suggest that long-term (evidenced by smoker vs. non-smoker comparisons) and short-term (acute) nicotine exposure may alter NMDA receptor-mediated arousal and mood systems in a way that promotes nicotine dependence in smokers, and addresses neurobiological deficiencies in smokers with schizophrenia.

Water exclusion at the nanometer scale provides long-term passivation of silicon (111) grafted with alkyl monolayers.

This work is a quantitative study of the conditions required for a long-term passivation of the interface silicon-alkyl monolayers prepared by thermal hydrosilyation of neat 1-alkenes on well-defined H-Si(111) surfaces. We present electrochemical capacitance measurements (C-U) in combination with ex situ atomic force microscopy (AFM) observations and X-ray photoelectron spectroscopy (XPS) measurements. Capacitance measurements as a function of the reaction time and XPS data reveal close correlations between the chemical composition at the interface and its electronic properties. A very low density of states is found if suboxide formation is carefully prevented. The monitoring of C-U plots and AFM imaging upon exposure of the sample in diverse conditions indicate that the initial electronic properties and structure of the interface are long-lasting only when the monolayer surface coverage is theta > 0.42. A model demonstrates that this threshold value corresponds to a monolayer with intermolecular channels narrower than approximately 2.82 A, which is equal to the diameter of a water molecule. Water exclusion from the monolayer promotes long-term passivation of the silicon surface against oxidation in air and water as well as perfect corrosion inhibition in 20% NH(4)F. We provide two criteria to assess when a sample is optimized: The first one is an effective dielectric constant <2.5, and the second one is a very characteristic energy diagram at open circuit potential.

Evaluation of the contribution of cyclooxygenase 1 and cyclooxygenase 2 to the production of PGE2 and PGF2 alpha in epithelial cells from bovine endometrium.

In ruminants, the production of prostaglandins by the endometrium is critical for recognition of pregnancy. In the absence of an embryonic signal, luteolytic pulses of PGF(2 alpha) are released by the uterus. In contrast, the presence of a viable conceptus reduces the production of PGF(2 alpha) relative to PGE(2) and prevents luteolysis through the release of trophoblastic interferon (IFN-tau). Initially, it was thought that epithelial and stromal endometrial cells were specialized in the production of a single type of prostaglandin. However, purified cell populations of both types of cell can produce PGF(2 alpha) and PGE(2); therefore, selective production of PGF(2 alpha) and PGE(2) must be regulated within each type of cell. Two distinct prostaglandin synthases, cyclooxygenase 1 and cyclooxygenase 2, are involved in prostaglandin production and each may catalyse the production of a different prostaglandin. This possibility was investigated in cultured epithelial cells from bovine endometrium. Cells were treated with oxytocin or arachidonic acid, and expression of cyclooxygenase 1 and cyclooxygenase 2 proteins was monitored over time and correlated with prostaglandin accumulation. Cells were also treated with increasing doses of inhibitors of cyclooxygenase 1 or cyclooxygenase 2 (non-steroidal anti-inflammatory drugs; NSAIDs) with or without arachidonic acid or oxytocin: flurbiprofen (0-50 micromol l(-1)) was used as a non-selective inhibitor; valeryl salicylate (0-500 micromol l(-1)) was used as a cyclooxygenase 1 inhibitor and NS-398 (0-1 micromol l(-1)) was used as a cyclooxygenase 2 inhibitor. After stimulation with arachidonic acid or oxytocin, prostaglandin production and expression of cyclooxygenase 2 protein were increased. All inhibitors were able to block basal and stimulated prostaglandin production. These results indicate that in endometrium most, if not all, prostaglandin production is probably processed through cyclooxygenase 2.

Weight loss, dysphagia, and outcome in advanced dementia.

There has been much debate on the value and risks of long-term enteral feeding in patients with advanced dementia. A retrospective study was carried out on 47 patients with a primary diagnosis of dementia who died over a two-year period. All were inpatients in a nursing home or skilled nursing facility. Marked weight loss and dysphagia occurring in a specific pattern were found to be associated with death from pneumonia. These clinical features probably imply failure of basic homeostatic mechanisms. Patients showing this clinical pattern may be less likely to show benefits from long-term enteral feeding.

Prevention concerning mental health: the adolescent's perspective.

OBJECTIVE: To examine several subjective components of adolescents' behaviour concerning mental illness prevention. METHOD: Adolescents' knowledge, their attitudes and subjective norms, as well as their thoughts about how they would concretely handle a psychological problem were measured. A self-administered questionnaire was completed by 438 male and female adolescents in grades 8 and 11. RESULTS: Gender and age differences were revealed: girls and older adolescents were more attuned to prevention. Further, the influence on young people of peers and parents was also apparent. CONCLUSION: Adolescents perceive prevention concerning mental health as important.

Trapping gel electrophoresis of end-labeled DNA: an analytical model for mobility and diffusion.

As shown by Ulanovsky, Drouin and Gilbert (Nature 1990, 343, 190-192), the gel electrophoretic migration of DNA is severely reduced by steric trapping when streptavidin is attached to one end of the polyelectrolyte. We present a model that allows us to calculate both the mobility and the diffusion coefficient, hence the resolution factor of the resulting separation. We compare our results to those of Défontaines and Viovy (Electrophoresis 1993, 14, 8-17) and we show that the averages over the molecular conformations must be done carefully. We also show that trapping increases diffusion substantially and that this makes constant-field trapping electrophoresis incapable of increasing the number of bases read per sequencing run. Finally, we conclude that severe trapping may lead to highly anomalous transport behavior where one cannot define a velocity or a diffusion constant.

Family therapy and some personality disorders in adolescence.

To prevent an endless debate between those supporting an intrinsic deficit versus an environmental deficit, the adolescent's problem has been seen here as both an individual problem and the metaphorical expression of a family problem. The intricacy of this two-faceted problem appears vividly with borderline and immature adolescents. Both the adolescent and family have, among other problems, difficulty separating. The approach described here focuses on that particular aspect; each therapeutic step is geared toward the adolescent's separation-individuation. Concerning the course of therapy, restructuring is perceived as the first therapeutic step and a prerequisite to work on such issues as delineating personal and family problems and improving communications. The approach is not, though, a panacea for the treatment of all personality disorders in adolescence and should not preempt the use of other approaches. As reported by Esman (1989) in regard to borderline adolescents, there is enough variation in the syndrome to allow the use of a variety of treatment approaches. Involving the family may not be feasible, and the adolescent sometimes must be removed from a detrimental family situation. The family of the immature or borderline adolescent is often seen as untreatable but what the therapist wants to accomplish and is ready to share often paves the way to effective intervention (Offer and Vanderstoep, 1974). The therapist must be ready to struggle with the family and with himself. The therapist has to be courageous, patient, and optimistic. The intervention is usually done on a middle-term basis with encouragement to the family to consult again if needed. The approach may thus appear obsolete at a time of a "quick-fix" culture. Family therapy can be a powerful tool with very disturbed, personality-disordered adolescents. The family approach must be flexible and based on a psychodynamic understanding of the problems. As so many of these cases fail to respond to the usual treatment and make therapists and institutions feel powerless, looking at the intervention from another angle can be refreshing. The experience can be rewarding for the adolescent, as she is becoming emancipated from the family, and for the parents, who facilitate the process. Working with these families is difficult but can also be rewarding for the clinician. Hours spent with them gives access to the subjective experiential reality that makes the substance of behavior and may allow the exploration of shadowy areas of the human condition. Family therapy becomes, then, a journey full of pain, uncertainties, and wonder for both the family and the clinician.

[Family therapy: what is at stake in its evolution and its present application]. / La thérapie familiale: les enjeux de son évolution et sa pratique actuelle.

Applying family therapy remains marginal in several child psychiatric clinics in spite of a favorable theoretical context. The author describes certain difficulties that the family therapist faces and places them into the context of the development of family therapy. Secondly, he shows the basic mechanisms that support this kind of intervention and the constant that is revealed in present practice. At the same time, the author offers a practical model allowing both the use of a psychoanalytical perspective and the systemic approach.

Psychological aspects of ultrasound imaging during pregnancy.

The psychological impact of ultrasound examination on expectant parents is assessed through direct observation, interviews and the administration of a questionnaire to a large group of parents. The examination was a positive and reassuring experience for most parents. Among a wide array of variables that could account for the effects of the exam, the results were the most important. Women viewing their first ultrasound, specially primiparae were more moved. The results did not confirm that there is a traumatic effect when viewing precedes quickening. Contrary to previous reports, fathers were as emotionally involved as the mothers. The presence of the father seemed also to have a beneficial effect on the mother. One-half of parents wanted to know the sex of the fetus before birth. These findings are discussed, along with implications for problems arising from introduction of new technologies during pregnancy.

[Evaluation of the family.]. / L'évaluation de la famille.

The author discusses the clinical family evaluation. He discusses its advantages, and difficulties, as well its dangers, and presents a model of family evaluation. Although developped with a view to a child psychiatry practice, this model could also be applied to other mental health intervention contexts. The model in question is based on family interactions and considers the developmental level of the children. In the course of the evaluation, the family is invited to accomplish some activities in which the children can participate. Clinical cases serve to illustrate the model. The family evaluation should facilitate a better understanding of the child's psychological problems.