Primer design to assess bacterial degradation of glyphosate and other phosphonates.

Phosphonates are organic phosphorous (P) compounds frequently detected in the environment due to a very stable CP bond that render them relatively recalcitrant. Glyphosate [N-phosphonomethyl glycine] is the most widely used and best-known synthetic phosphonate, and one of the most concerning herbicides in the world today. Microbial degradation of glyphosate and organophosphonates in general, is the main dissipation mechanism operating in most environments. One microbial metabolic pathway in this process is the CP lyase pathway, entailing an enzymatic complex encoded by about 14 genes (the Phn operon). Our goal was to develop a quantitative polymerase chain reaction (qPCR) assay for a key enzyme, the CP lyase that breaks down the CP bond, via quantification of the codifying phnJ gene. The primers designed in this study fulfill the requirements for a successful qPCR assay, with high efficiency and sensitivity, as well as specific detection of the target sequence in a wide range of taxonomic groups. This is, to our knowledge, the first report of primers designed to target phnJ in both pure cultures and metagenomic DNA from different environmental sources. Direct quantification of phnJ may be a cost-effective proxy to determine glyphosate degradation potential in different matrixes.

CD4 and CD4/CD8 ratio progression in HIV-HCV infected patients after achievement of SVR.

BACKGROUND: In HIV-HCV co-infected patients, the long-term effects of HCV eradication on HIV disease progression are still unclear. OBJECTIVES: This study aims to determine if CD4 and CD4/CD8 ratio slopes improved after anti-HCV treatment in patients achieving a sustained virological response (SVR). STUDY DESIGN: A total of 116 HIV-HCV co-infected patients, previously treated with Peg-IFN/RBV, were divided into two groups: SVR (55 patients who had achieved SVR), and non-SVR (61 patients). Retrospective data before and after anti-HCV therapy were obtained for all patients, with a median 8 year-follow-up. Multilevel mixed models were fitted to assess the trends over time of FIB-4 score, APRI score, CD4, CD8 cell count and CD4/CD8 ratio. RESULTS: Median HIV-infection duration, HCV-RNA and GGT baseline levels were higher in non-SVR compared to the SVR group. A significantly decreased FIB-4 (p<0.001) and APRI trend (p<0.001) after SVR was observed in SVR patients compared to those non-SVR. After adjustment for HIV duration, there was no significant difference between the two groups for absolute CD4 (p=0.08) or percentage CD4 slope (p=0.6) over time. The CD4/CD8 ratio trend also demonstrated a similar progressive increase in both groups (p=0.2). During follow-up, six deaths were reported in the non-SVR group versus no death for the SVR group, while no difference in AIDS and non-AIDS events was observed. CONCLUSIONS: Achievement of SVR determines an important beneficial impact in terms of liver-related mortality and fibrosis regression, but does not seem to alter neither the slope of long term CD4 gain nor the CD4/CD8 ratio evolution in ART-treated HIV-HCV co-infected patients.

Ion microscopy based on laser-cooled cesium atoms.

We demonstrate a prototype of a Focused Ion Beam machine based on the ionization of a laser-cooled cesium beam and adapted for imaging and modifying different surfaces in the few-tens nanometer range. Efficient atomic ionization is obtained by laser promoting ground-state atoms into a target excited Rydberg state, then field-ionizing them in an electric field gradient. The method allows obtaining ion currents up to 130pA. Comparison with the standard direct photo-ionization of the atomic beam shows, in our conditions, a 40-times larger ion yield. Preliminary imaging results at ion energies in the 1-5keV range are obtained with a resolution around 40nm, in the present version of the prototype. Our ion beam is expected to be extremely monochromatic, with an energy spread of the order of the eV, offering great prospects for lithography, imaging and surface analysis.

Pure optical nano-writing on light- switchable spiropyrans/merocyanine thin film.

We report optical writing at the nanometer scale of spin coated PMMA-spiropyran films. By using a near-field optical microscope, pure optical nano-writing with a resolution of 160 nm and writing speed of 0.4 µm/s was achieved. Simultaneous topographic and optical writing was also obtained by simply coupling to the near-field few more mW of laser power. Due to the fast optical response of the spiropyran molecule, nano-lithography on PMMA-spyropyran thin films appears to be very attractive for future photonics applications.

Rovibrational cooling of molecules by optical pumping.

We demonstrate rotational and vibrational cooling of cesium dimers by optical pumping techniques. We use two laser sources exciting all the populated rovibrational states, except a target state that thus behaves like a dark state where molecules pile up thanks to absorption-spontaneous emission cycles. We are able to accumulate photoassociated cold Cs(2) molecules in their absolute ground state (v = 0, J = 0) with up to 40% efficiency. Given its simplicity, the method could be extended to other molecules and molecular beams. It also opens up general perspectives in laser cooling the external degrees of freedom of molecules.

Deeply bound cold caesium molecules formed after 0(-)(g) resonant coupling.

Translationally cold caesium molecules are created by photoassociation below the 6s + 6p(1/2) excited state and selectively detected by resonance enhanced two photon ionization (RE2PI). A series of excited vibrational levels belonging to the 0(-)(g) symmetry is identified. The regular progression of the vibrational spacings and of the rotational constants of the 0(-)(g) (6s + 6p(1/2)) levels is strongly altered in two energy domains. These deviations are interpreted in terms of resonant coupling with deeply bound energy levels of two upper 0(-)(g) states dissociating into the 6s + 6p(3/2) and 6s + 5d(3/2) asymptotes. A theoretical model is proposed to explain the coupling and a quantum defect analysis of the perturbed level position is performed. Moreover, the resonant coupling changes dramatically the spontaneous decay products of the photoexcited molecules, strongly enhancing the decay into deeply bound levels of the a(3)Σ(+)(u) triplet state and of the X(1)Σ(+)(g) ground state. These results may be relevant when conceiving population transferring schemes in cold molecule systems.

Comparative animal study on hard tissue integration and bone formation of different Nobel Biocare implants.

Dental implantation aims at optimal and long-term hard tissue integration. Beside primary stability, loading time and other factors, e.g. the surface of the endosteal part of the implant, is a matter of special importance. In this animal trial, hard tissue integration of two different implant types was studied using radiological, histological and histomorphometric analysis. Two different implants with an oxidized surface (TiUnite; Nobel Biocare AB, Goteborg, Sweden, NobelReplace Tapered Groovy 4.3 x 10 mm and Replace Select Tapered 4.3 x 10 mm) were inserted into the right and left mandibles of 10 German domestic pigs between canine and premolar and immediately provided with a ceramic crown. The primary implant stability was determined using resonance frequency analysis. After 70 days, the test animals were killed and specimens were collected for histological and histomorphometric examination. All implants showed good primary stability after surgery. Histological and histomorphometrical analysis revealed no significant differences in the bone apposition. The immediate loading of the different implant types don't have any negative effects on the bone apposition in the period of 70 days. The long-term effects of immediate loading of these types of implant requires further studies.

Soft tissue integration in the neck area of titanium implants--an animal trial.

Dental implant materials are required to enable good apposition of bone and soft tissues. They must show sufficient resistance to chemical, physical and biological stress in the oral cavity to achieve good long-term outcomes. A critical issue is the apposition of the soft tissues, as they have provided a quasi-physiological closure of oral cavity. The present experiment was performed to study the peri-implant tissue response to non-submerged (1-stage) implant installation procedures. Two different implants types (NobelBiocare, NobelReplace Tapered Groovy 4.3 x 10 mm and Replace Select Tapered TiU RP 4.3 x 10mm) were inserted into the right and left sides of 8 domestic pigs (Sus scrofa domestica) mandibles, between canines and premolars and immediately provided with a ceramic crown. Primary implant stability was determined using ressonance frequency analysis. Soft tissue parameters were assessed: sulcus depth (SDI) and junctional epithelium (JE). Following 70 days of healing, jaw sections were processed for histology and histomorphometric examination. Undecalcified histological sections demonstrated osseointegration with direct bone contact. The soft tissue parameters revealed no significant differences between the two implant types. The peri-implant soft tissues appear to behave similarly in both implant types.

Balanced homodyning for apertureless near-field optical imaging.

A quadrature optical detection technique, based on polarized balanced-homodyne interferometry, has been developed for specific application to apertureless near-field scanning optical microscopy (ANSOM). With such technique, multiplicative background interference, inficiating quantitative optical imaging in standard homodyne-based ANSOM, can be suppressed. Periodic modulation of interferometric optical phase, typically employed in heterodyne-based ANSOMs even to such purpose, is not needed in the present configuration. Homodyne detection also facilitates detection of harmonic components of the ANSOM optical signal at the probe/sample distance modulation frequency, necessary for near-field discrimination and suppression of artifacts. Furthermore, since amplitude signal is not affected by phase fluctuations generated in the optical path of the interferometer, an optical fiber could be included in one interferometer arm, to couple the ANSOM head to the detection system, obtaining improved versatility of the instrument. A demonstration of the interferometer performance is given by a test confocal optical scan of a mirror surface. This technique, as applied to near-field microscopy, is anticipated to provide absolute values of optical contrast not depending on background interference and topography artifacts.

Mapping local field enhancements at nanostructured metal surfaces by second-harmonic generation induced in the near field.

We report on an aperture scanning near-field optical microscope in which femtosecond pulses are coupled to a hollow-pyramid aperture sensor. Such probe displays high throughput and preserves pulse duration and polarization, enabling the achievement of sufficiently high peak power in the near field to perform nonlinear optics on the nanoscale. We use the system to observe the nonlinear optical response of nanostructured metal surfaces with sub-100-nm spatial resolution. We study second-harmonic generation from gold nanoparticles both isolated and in high-density patterns, highlighting a strong dependence of the generation efficiency on the shape and on the fine structure of the nanoemitter. In particular, we present results on closely packed gold triangles as well as on nanoellipsoids with different local surface plasmon resonances.

Real-time monitoring of the surface relief formation on azo-polymer films upon near-field excitation.

We use near-field optical microscopy to investigate the early state formation of photo-induced topographical structures on the free surface of azobenzene-containing polymers. The near-field technique is employed to monitor in real time the mass migration during the embossing of the surface of 50/50 copolymer films of polymethacrylate and polymethacrylate containing 3-methyl-4'-pentyloxy-azobenzene units. The dynamic of surface relief formation induced by light have been investigated as a function of the sample temperature. Furthermore, a formula derived from Navier-Stokes equations for classical laminar flow has been used to fit the mass displacement data during the embossing process. Excellent agreements between the experimental data and the fitting formula have been found for all the temperature considered.

Polarization-maintaining near-field optical probes.

We demonstrate that tapered optical fibre probes can be easily modified in the taper cone to realize an electric dipole producing a well-defined near-field polarized light. This novel structure is made of a Short-cut Double C-shaped probe design combined to the usual full metal coating near the tapered end of the fibre. Hence, the cone at the apex of the probe is excited by an equivalent dipole whose spatial orientation is dictated by the probe geometry, regardless the polarization state of the incoming light. Properties and performances of such a configuration are first predicted by a finite-difference time domain simulation, showing that the near field coming out from the probe is linearly polarized. Following this novel design, a probe prototype is manufactured and tested. Its measured polar diagram confirms the polarization maintenance property in the near field.

Wide angle near-field optical probes by reverse tube etching.

We present a simple modification of the tube etching process for the fabrication of fiber probes for near-field optical microscopy. It increases the taper angle of the probe by a factor of two. The novelty is that the fiber is immersed in hydrofluoric acid and chemically etched in an upside-down geometry. The tip formation occurs inside the micrometer tube cavity formed by the polymeric jacket. By applying this approach, called reverse tube etching, to multimode fibers with 200/250 microm core/cladding diameter, we have fabricated tapered regions featuring high surface smoothness and average cone angles of approximately 30 degrees . A simple model based on the crucial role of the gravity in removing the etching products, explains the tip formation process.

Highly efficient second-harmonic nanosource for near-field optics and microscopy.

A nanometric source of second-harmonic (SH) light with unprecedented efficiency is demonstrated; it exploits the grazing-incidence illumination of a metal tip, which is conventionally used for atomic force microscopy, by 25-fs laser pulses of a high-energy Ti:sapphire oscillator. Tip scanning around the beam focus shows that the SH generation is strongly localized at its apex. The polarization dependence of the SH light complies with the model of an on-axis nonlinear oscillating dipole.

A versatile multipurpose scanning probe microscope.

A combined scanning probe microscope has been developed that allows simultaneous operation as a non-contact/tapping mode atomic force microscope, a scattering near-field optical microscope, and a scanning tunnelling microscope on conductive samples. The instrument is based on a commercial optical microscope. It operates with etched tungsten tips and exploits a tuning fork detection system for tip/sample distance control. The system has been tested on a p-doped silicon substrate with aluminium depositions, being able to discriminate the two materials by the electrical and optical images with a lateral resolution of 130 nm.

Near-field Raman imaging of morphological and chemical defects in organic crystals with subdiffraction resolution.

In this study we report on the application of an aperture near-field optical microscope for Raman imaging of organic materials. Spectral analysis and detailed Raman imaging are performed with integration times of 100 ms per point, without the aid of field enhancement effects. The studied samples consist of two high Raman-efficiency molecular samples: a 7,7',8,8'-tetracyanoquinodimethane crystal showing surface defects and a 7,7',8,8'-tetracyanoquinodimethane thin film characterized by the presence of submicrometre-sized organometallic copper-salt complexes. In the first case, the effect of the surface deformation was studied, whereas in the second sample we were able to chemically image the formation of salt complexes. Subdiffraction resolution was achieved in both studies.

Morphologic variations in bacteria under stress conditions: near-field optical studies.

The morphologic and structural variations suffered by cells of a population of Pseudomonas aeruginosa ATCC 27853 under stress conditions were investigated by using scanning near-field optical microscopy. The analysis of the images, supported by microbiological data, showed that the bacteria evolved from the initial distribution of rod-shaped cells of standard size to a population with structural and morphologic modifications. The detection of variations in the optical reflectivity over a subwavelength scale (< or = 100 nm), combined with the concurrently acquired topographical signal, allowed the visualisation of rod-shaped bacteria going towards a lytic process and entire "U"-shaped cells. In the latter cells, which derived from a morphology refolding of rod bacteria, cellular matter seemed to rearrange itself to attain a coccoid stress resistant form, responsible for the residual viability of the population.

Optical near-field harmonic demodulation in apertureless microscopy.

Spatial derivatives of the optical fields scattered by a surface can be investigated by apertureless near-field optical microscopy by modulating sinusoidally the probe to sample distance and detecting the optical signal at the first and higher harmonics. Demodulation up to the fifth harmonic order has been accomplished on a sample of close-packed latex spheres by means of the silicon tip of a scanning interference apertureless microscope. The working principles of such microscope are reviewed. The experimental configuration used comprises a tuning-fork-based tapping-mode atomic force microscope for the distance stabilization, and a double-modulation technique for complete separation of the topography tracking from the optical detection. Simple modelling provides first indications for the interpretation of experimental data. The technique described here provides either artefact-free near-field optical imaging, or detailed information on the structure of the near fields scattered by a surface.

Photoassociative spectroscopy as a self-sufficient tool for the determination of the Cs triplet scattering length

In photoassociation spectroscopy, the line intensities of a given vibrational progression exhibit zero-signal modulation reflecting the node structure of the s-wave ground state wave function of two free colliding atoms. This leads to the determination of the scattering length. We performed photoassociation of cold Cs atoms polarized in the Zeeman sublevel f = 4, m(f) = 4. We analyzed the intensities of the lines associated with the Cs2 0(-)(g) state dissociating to the 6s(1/2)+6p(3/2) asymptote. This yields a value of the Cs triplet state scattering length, a(T) = -530a(0), while consistency requirements impose a value of the multipole ground state molecular coefficient, C6 = 6510 a.u.