Article history: Available online 9 August 2010

La biologie moleculaire est longtemps restee une discipline depourvue de solutions d'automatisation. Toutefois, depuis le debut des annees 2000, les efforts conduits par les industriels du domaine ont contribue au developpement d'une offre instrumentale etoffee et a meme de satisfaire un large nombre d'utilisateurs. Le lancement sur le marche d'instruments automatisant l'extraction des acides nucleiques et l'automatisation de la PCR temps reel associe a la disponibilite de differents kits, par exemple dans le domaine infectieux, contribue a une augmentation tangible du nombre de laboratoires realisant des analyses de biologie moleculaire. Nous presentons dans cet article trois tableaux donnant un apercu synthetique (informations compilees en Juin 2007) de l'offre disponible dans les domaines de l'extraction des acides nucleiques et de la PCR temps reel. Les grandes tendances de ce marche sont egalement abordees.

On parle depuis longtemps de l’utilisation des biopuces en biologie moléculaire, mais ce type d’instrument n’est pour l’instant, que peu utilisé dans les laboratoires de diagnostic médical. Les technologies disponibles et leur coût y sont pour beaucoup. Leurs applications disponibles et leur adéquation aux demandes diagnostic pur sont deux autres éléments de réponse. Pourtant, cette tendance pourrait changer avec l’apparition de solutions technologiques plus simples, peu onéreuses et ciblées sur les demandes d’analyses d’aujourd’hui. Les instruments provenant de sociétés comme Greiner Bio-One, Clondiag, Osmetech, Nanogen, Autogenomics, Innogenetics et Nanosphere donnent des indications sur ces nouvelles approches qui pourraient bien susciter un nouvel intérêt des biologistes, avec en particulier l’arrivée des nanotechnologies sur nos paillasses.

ABSTRACTIn this study, named the Zephir study (Telzir-pharmacokinetics), 121 antiretroviral-experienced human immunodeficiency virus (HIV) patients failing on highly active antiretroviral therapy (HAART) were included in a prospective cohort and received a fosamprenavir-ritonavir (700 mg/100 mg twice a day)-based regimen. The impact of baseline HIV type 1 (HIV-1) mutations, pharmacokinetic (PK) parameters, and genotype inhibitory quotient (GIQ) on the virological response at week 12 (W12) was assessed. HIV reverse transcriptase and protease were sequenced at W0. The response at W12 was defined as <2.3 log10HIV-1 RNA copies/ml or a virus load decrease of ≥1 log10copies/ml. W4 amprenavir PK were determined by high-performance liquid chromatography. Patients had a median of nine previous treatments over 8 years. Median W0 values were as follows: 295 CD4+/μl, 4.4 log10HIV-1 RNA copies/ml, and 6 protease- and 5 nucleotide reverse transcription inhibitor-related mutations. Respective values for minimum concentration of drug in serum (Cmin) and area under the concentration-time curve (AUC) from 0 to 24 h were 1,400 ng/ml and 35 mg·h/ml. At W12, 52% of the patients were successes, with a median decrease of −0.7 log10HIV-1 RNA copies/ml. The Zephir mutation score included 12 IAS protease mutations associated with poorer virological response: L10I/F/R/V, L33F, M36I, M46I/L, I54L/M/T/V, I62V, L63P, A71I/L/V/T, G73A/C/F/T, V82A/F/S/T, I84V, L90M, and polymorphism mutations I13V, L19I, K55R, and L89M. Comparing <4 versus ≥4 mutations, HIV-1 RNA decreases were −2.3 log10copies/ml versus −0.1 log10copies/ml (P< 10−4) with 93% versus 19% successes (P< 10−4), respectively. This score predicted W12 failure with 94% sensitivity, versus 31% for the ANRS 2005 algorithm.Cmin(<1,600 ng/ml), AUC (<40 mg·h/ml), and GIQ (<300) values were associated with failure (allPvalues were <10−4). The need to test genotype-based algorithms using different patient databases before their implementation in clinical practice is highlighted. Specific mutations, PK and GIQ, provide relevant information for monitoring fosamprenavir-ritonavir-based HAART.

Thin films of vanadium oxide were prepared and studied for the electro-optical properties of semiconductor-metal transition. Vanadium oxide films with thickness of ~ 0.15μm were deposited on SiO2/Si substrates by reactive RF magnetron sputtering using a pure vanadium target under various ratios of argon and oxygen gases. The oxygen content in the mixed atmosphere has a significant influence on the semiconductor-metal transition characteristics. Both thermochromic and electrochromic modes were studied. In thermochromic mode, the oxide film deposited in an O2/Ar ratio of 1.2% exhibits 90% optical transmission in semiconducting state at room temperatures, while very low transmission at 5% in metallic state at 65°C, in the wavelength region of 8 to 12μm. In the near infrared region of 1 to 2μm, the transmission is about 60% in the semiconducting state and a few percents in the metal state. A corresponding three-order variation of resistivity was observed over the transition. The refractive indices (n and k) of the vanadium oxide films were measured using an ellipsometer in the near infrared region between 1 and 2 μm in both states. The index n decreases in metal state while k increases. The electrochromic phase transition of vanadium oxide was investigated by applying a pulsed voltage to minimize the heating effect. The required charge density for the phase transition is consistence with the Mott metal-insulator model. Longwave IR switching and modulation were demonstrated by electrically induced semiconductor-metal transition.

This paper consists of an investigation of the structural arrangement of the sp2 phase in amorphous unhydrogenated carbon nitride (a-CNx) films and its effect on their physical properties. The a-CNx films (0.161020∕cm3) and the EPR linewidth (of a few gausses) along with a decrease in nitrogen content. Visible Raman measurements indicate that these effects are accompanied by an increase in the degree of disorder of the sp2 phase, as inferred from the broadening and downshift of the G Raman band, and a reduction of the CN triple bond signal. The analysis of these results in terms of the structural configuration and bonding in the films, show that an enhancement of the connectivity of the sp2 phase in the layers, takes place when deposition is performed at high laser intensities. These structural modifications are strongly correlated to a decrease in the optical gap from 0.61 to 0.21 eV as well as to an increase of the hardness value of the films from 12 to 24 GPa. The transition from a reduced to an enhanced connectivity of the sp2 phase occurs when the nitrogen content decreases below 22 at. %, as a result of the detected reduction of the triply bonded CN species in the layers.

Silicon nitride microbridges (50x50 mu m(2), 0.6 mu m thick), suspended over a silicon substrate, were patterned and thinned. These patterns consist of 2 to 12 windows that were thinned to approximately 0.3 mu m. Microbolometers were fabricated by sputtering a YBaCuO thin film over the bridges. The experimental results showed that the regionally thinned microbridges have a lower thermal time constants tau(about 1.6 ms) than that of the standard pixel configuration (2.6 ms). On the other hand, the fact that the regionally thinned microbolometers having detectivity D* values comparable to or even six times superior than that of the standard pixel showed that the decrease in response time is not penalized by loss of detection performance. The simulation results also show that as the amount of material removed is increased, the thermal time constant drops significantly while the (tau/G)(1/2) ratio (where G is the thermal conductance of the pixel) only decreases slightly, suggesting that the reduced response time will not cause a significant drop in detectivity D*. The simulation results of mechanical integrity show that a specific regionally thinned microbridge design has 22% higher stiffness than that of a standard pixel design with similar thermal properties. The fact that thick regions remained on the regionally thinned pixels (like the edges of the pixels) provide significant mechanical support to the microstructures. This confirms the validity of the regionally thinned microbridges approach.

After a lively discussion at LabAutomation2005 in San Jose, we asked three major diagnostic companies to share their vision for the molecular diagnostics “rapid-growing-and-more-than-ever” market. Happily, all of them complied and we are pleased to present insight from Bayer Healthcare, Beckman Coulter, and Roche Molecular Systems. To complement these perspectives, we invited a heavy user of molecular diagnostic assays to comment on today's situation and share his hopes for the future of molecular medicine. It is with pleasure that we feature insight from Philippe Halfon of Alphabio Laboratoire.

Because the effects of treatment interruption on patients with >400 CD4(+) cells/microl and prolonged moderate HIV loads is unknown, their HIV1 RNA and DNA loads, plasma, and PBMC resistance mutations and CD4+ kinetics were studied during 12 months of treatment interruption. Fifty-seven patients were included: 28 with undetectable (median 40 months) and 29 with moderate (>1 year) HIV1 RNA levels (3.3 log10 copies/ml) at the baseline M0. HIV1 RNA and CD4+ counts were determined monthly. PBMC HIV1 DNA was quantified (real-time PCR) and its reverse transcriptase (RT) and protease (PR) genotypes analyzed (M0, M6, M12). Regardless of HIV1 RNA detectability at the baseline M0, all patients had comparable HIV1 RNA (median 4.6), DNA (median 2.9), CD4+ (median 455) at month 12 (M12). The decisive moment was month 1 (M1), when the HIV1 RNA increase and CD4 decline stabilized, rendering M0 differences non-significant and predicting outcome. The lower the CD4+ nadir was before HAART, the steeper the CD4+ decline at M1. HIV1 DNA shifted to wild-type genotype in 47% (M6) and 79% (M12) of RT; 64% of PR major resistance mutations disappeared (M12). Treatment interruption is possible, regardless of the baseline M0 HIV RNA status, but it must take into consideration the CD4+ nadir, an outcome predictor, to initiate HAART before too severe depletion to preserve the possibility of treatment interruption. HIV1 DNA genotyping helps monitor patients with undetectable HIV RNA at M0.

We present an investigation of the effect of the process parameters, namely deposition pressure and laser intensity, on the growth and mechanical properties of carbon nitride (CNx) thin films synthesized by plasma assisted pulsed laser deposition. Deposition at high remote plasma pressure (200 mTorr) enhances both growth rate and nitrogen incorporation (up to 40 at. %), but nano-indentation measurements indicate that these films are very soft and have poor mechanical properties. At low remote plasma pressure (0.5 mTorr), the nitrogen content varies from 24 to 16 at. % with increasing laser intensity as the films become much harder and more elastic, with hardness and Young's modulus values reaching 24 GPa and 230 GPa, respectively. These effects are explained in terms of a thermalization of the laser plasma at 200 mTorr and indicate that plasma activation of nitrogen does not provide any particular benefit to the film properties when deposition is performed at high pressure. However, at low pressure, the benefit of plasma activation is evidenced through enhanced nitrogen incorporation in the films while preserving the highly energetic species in the ablation plume. Such conditions lead to the synthesis, at room temperature, of hard and elastic films having properties close to those of fullerene-like CNx.

In this work, Raman spectroscopy and electron paramagnetic resonance (EPR) are used to investigate sp2 carbon atoms nanoclustering in both diamond-like-carbon (DLC) and amorphous carbon nitride (CNx) films grown at room temperature by pulsed laser deposition. For DLC, ablation of a graphite target using a KrF laser was performed under vacuum, at different laser intensities. CNx growth was achieved using the same system to which a nitrogen atom source was added. For both types of films, increasing the laser intensity was found to reduce the size of the sp2 nanoclusters, as deduced from the variation of the D to the G Raman peaks intensity ratio. Moreover, the estimated size of the sp2 nanoclusters in CNx appears to be consistently larger than that in DLC films. The Lorentzian EPR signal, with a g-value of 2.0028 and room temperature linewidth ranging from approximately 2.3 to 3.9 G, gives further indication about clustering of sp2 paramagnetic centers (with measured spin densities >1020/cm3). For all films, the EPR linewidth was found to narrow down to approximately 2 G, when measured at 80 K. The EPR line broadening with increasing temperature is greater for the CNx films. Possible structural configurations of the nanoclusters, depending on laser intensity and nitrogen incorporation, are discussed.

Carbon nitride thin films have been deposited at room temperature by plasma assisted pulsed laser deposition and the effect of varying the KrF excimer laser intensity was studied by examination of the bonding and hardness of the films. X-ray photoelectron spectroscopy shows that the nitrogen content in the films decreases from 24 to 16 at.% when laser intensity is increased from 2.5×107 to 9.4×108 W/cm2 and that the nitrogen atoms are incorporated in the films under two different bonding configurations, as deduced by two distinct contributions to the N 1s signal at 398.4±0.2 and 400.1±0.2 eV. However, it was found that the stoichiometry of the CN bonded structures is independent of laser intensity as it remains constant at 33±3 at.% of N. This suggests that the microstructure of the films consists of a nearly stoichiometric C2N phase mixed with amorphous carbon regions. Such a picture is further supported by the observation that the binding energy of the two components of the N 1s XPS signal is not affected by laser intensity. By using continuous stiffness measurement nanoindentation, a hardness value of 24 GPa, was determined for the film synthesized at the highest laser intensity used in this work but it was found to deteriorate with increasing nitrogen content. These effects could be explained in terms of an intermixing between the C2N phase and pure carbon domains rather than a modification of nitrogen bonding configuration in the films.

Of 75 human immunodeficiency virus (HIV) type 1-infected patients successfully responding to 2 nucleoside reverse-transcriptase inhibitors (NRTIs) plus 1 protease inhibitor (PI), 55 started a simplified abacavir (ABC)-based triple NRTI regimen. Influences of DNA load and DNA reverse-transcriptase (RT) mutations on virological responses were assessed at month 6 after initiation of therapy. Baseline heterogeneity was observed: peripheral blood mononuclear cell (PBMC) genotyping showed 31% RT mutations with 1-5 NRTI-related mutations, 78% protease mutations had 1-5 PI-related mutations; and HIV-1-DNA levels were 1.8-3.5 log(10) copies/10(6) PBMC. Outcomes for 49 patients on a regimen of 2 NRTIs plus ABC were as follows: 22 successes, 10 blips ("blip" defined as intermittent plasma HIV-1 RNA levels between 50 and 100 copies/mL and a return to an undetectable level), and 17 failures, whereas, for patients continuing on a regimen of 2 NRTIs plus 1 PI, there were 19 successes and 1 blip. Previous treatment regimens, baseline provirus level, and PBMC genotype predicted virological outcome.

Le sequencage des acides nucleiques est une technique qui a ete a la base du developpement de la biologie moleculaire des le debut des annees 70. Des instruments permettant ce genre d'analyse se sont rapidement developpes dans les annees 80 et 90. Depuis la fin des annees 90, de nouvelles technologies de sequencage sont apparues pour de nouvelles generations de sequenceurs automatiques. A cote des sequenceurs en gel plat, les sequenceurs capillaires ont apporte une plus grande automatisation dans les laboratoires de plus en plus demandeurs en sequencage de routine. Cependant de nouvelles procedures font toujours leur apparition. Le pyrosequencage, la spectrometrie de masse, la microfluidique et les puces a ADN, font partie de ces technologies qui feront vraisemblablement avancer l'instrumentation de sequencage dans les prochaines annees. En attendant, on trouvera dans le tableau propose, l'instrumentation disponible sur le marche francais pour le sequencage des acides nucleiques.

Le sequencage des acides nucleiques est une technique qui a ete a la base du developpement de la biologie moleculaire des le debut des annees 70. Des instruments permettant ce genre d'analyse se sont rapidement developpes dans les annees 80 et 90. Depuis la fin des annees 90, de nouvelles technologies de sequencage sont apparues pour de nouvelles generations de sequenceurs automatiques. A cote des sequenceurs en gel plat, les sequenceurs capillaires ont apporte une plus grande automatisation dans les laboratoires de plus en plus demandeurs en sequencage de routine. Cependant de nouvelles procedures font toujours leur apparition. Le pyrosequencage, la spectrometrie de masse, la microfluidique et les puces a ADN, font partie de ces technologies qui feront vraisemblablement avancer l'instrumentation de sequencage dans les prochaines annees. En attendant, on trouvera dans le tableau propose, l'instrumentation disponible sur le marche francais pour le sequencage des acides nucleiques.

To date the majority of sequencing technologies have been based on use of gel plates. In this study sequencing by capillary electrophoresis for HIV-1 genotyping on the CEQ 2000 sequencer (Beckman Coulter Inc.) has been investigated and compared to an ‘in house’ protocol on the Prism-377 sequencer (Applied Biosystems) and to the HIV-1 TruGene kit (Visible Genetics Inc.), two gel plate-based systems. Plasma from 20 HAART-treated patients with virological failure were analyzed for protease (PR) and reverse transcriptase (RT) genes. A total of 520 RT codons (26/patient) and 360 PR codons (18/patient) related to antiretroviral drug resistance were evaluated. The overall agreement between CEQ 2000 and Prism-377 results was 100% for the RT and PR primary and secondary mutations. The overall agreement between CEQ 2000 and TruGene was 100% for primary and≥97% for secondary mutations. Discrepant results would have never led to errors in genotype interpretation. Performances for a 24 patients/week/one technician genotyping throughput were analyzed. For Prism-377, TruGene and CEQ 2000, manual processing required 5, 4 and 2,5 days, sequence data analysis needed additional 3, 1 and 2 days and cost/patient was≈49, 214 and 39 $, respectively. The CEQ 2000 sequencer offers a reliable alternative for fast and cost effective HIV drug resistance analysis.