Two-dimensional structured illumination microscopy.

In widefield fluorescence microscopy, images from all but very flat samples suffer from fluorescence emission from layers above or below the focal plane of the objective lens. Structured illumination microscopy provides an elegant approach to eliminate this unwanted image contribution. To this end a line grid is projected onto the sample and phase images are taken at different positions of the line grid. Using suitable algorithms 'quasi-confocal images' can be derived from a given number of such phase-images. Here, we present an alternative structured illumination microscopy approach, which employs two-dimensional patterns instead of a one-dimensional one. While in one-dimensional structured illumination microscopy the patterns are shifted orthogonally to the pattern orientation, in our two-dimensional approach it is shifted at a single, pattern-dependent angle, yet it already achieves an isotropic power spectral density with this unidirectional shift, which otherwise would require a combination of pattern-shift and -rotation. Moreover, our two-dimensional approach also yields a better signal-to-noise ratio in the evaluated image.

Long-term efficacy and safety of combined prolonged-release oxycodone and naloxone in the management of non-cancer chronic pain.

OBJECTIVE: The aim of this study was to assess safety and efficacy of fixed combination oxycodone prolonged release (PR)/naloxone PR in terms of both analgesia and improving opioid-induced bowel dysfunction (OIBD) and associated symptoms, such as opioid-induced constipation (OIC), in adults with chronic non-cancer pain. STUDY DESIGN: These were open-label extension studies in which patients who had previously completed a 12-week, double-blind study received oxycodone PR/naloxone PR for up to 52 weeks. The analgesia study assessed pain using the modified Brief Pain Inventory-Short Form (BPI-SF). The bowel function study assessed improvements in constipation using the Bowel Function Index (BFI). RESULTS: At open-label baseline in the analgesia study (n = 379), mean score [+/- standard deviation (SD)] for the BPI-SF item 'average pain over the last 24 h' was 3.9 +/- 1.52, and this remained low at 6 months (3.7 +/- 1.59) and 12 months (3.8 +/- 1.72). Mean scores for BPI-SF item 'sleep interference', and the BPI-SF 'pain' and 'interference with activities' subscales also remained low throughout the 52-week study. In the bowel function study (n = 258), mean BFI score (+/- SD) decreased from 35.6 +/- 27.74 at the start of the extension study to 20.6 +/- 24.01 after 12 months of treatment with oxycodone PR/naloxone PR. Pain scores also remained low and stable during this study. Adverse events in both extension phases were consistent with those associated with opioid therapy; no additional safety concerns were observed. CONCLUSION: Results from these two open-label extension studies demonstrate the long-term efficacy and tolerability of fixed combination oxycodone PR/naloxone PR in the treatment of chronic pain. Patients experienced clinically relevant improvements in OIBD while receiving effective analgesic therapy.

Combined prolonged-release oxycodone and naloxone improves bowel function in patients receiving opioids for moderate-to-severe non-malignant chronic pain: a randomised controlled trial.

BACKGROUND: This randomised, double-blind, double-dummy, parallel-group multicentre study assessed the impact of a total daily dose of 60-80 mg oral oxycodone prolonged-release (PR)/naloxone PR (OXN PR) as fixed-ratio combination for patients with opioid-induced constipation (OIC) having moderate-to-severe, non-malignant pain. METHODS: During pre-randomisation patients receiving opioids for moderate-to-severe non-malignant pain were converted to oxycodone PR (OXY PR) and titrated to an effective analgesic dose. During randomisation 265 patients on a stable OXY PR dose (60-80 mg/day) and with OIC were included in the full analysis population to receive OXN PR or OXY PR alone. Primary outcome was improvement in symptoms of constipation as measured by the Bowel Function Index (BFI). Secondary/exploratory outcomes examined analgesic efficacy and other bowel function parameters. RESULTS: After 4 weeks of treatment, patients receiving OXN PR showed a significant improvement in bowel function compared with those in the OXY PR group (-14.9; 95% CI: -17.9, -11.9; p<0.0001) as measured by BFI which was seen after only 1 week of treatment continuing to the end of the study. After 4 weeks of treatment, patients receiving OXN PR had a median number of 3.0 complete spontaneous bowel movements (CSBM) per week compared with only 1.0 for OXY PR alone. Laxative intake was lower in the OXN PR than the OXY PR group. Furthermore, improvements in bowel function were achieved without loss of analgesic efficacy; pain intensity scores were comparable between the groups and consistent for duration of the study. Most frequently reported adverse events were consistent with those reported for opioid analgesics; no new or unexpected adverse reactions attributable to OXN PR used in higher doses were observed. CONCLUSION: This study shows that the fixed-ratio combination of OXN PR is superior to OXY PR alone in terms of bowel function, while providing effective equivalent analgesia.

Anti-inflammatory properties of a liposomal hydrogel with povidone-iodine (Repithel) for wound healing in vitro.

A liposomal hydrogel with 3% povidone-iodine (PVP-ILH, Repithel) has shown clinical benefit in settings where inflammation and/or reactive oxygen species are thought to impede wound healing (e.g., burns, chronic wounds and in smokers). This in vitro study investigated whether PVP-ILH is able to reduce inflammatory events responsible for the impairment of the wound healing process in such patients. Therefore, the following assays were conducted with PVP-ILH (and derived control hydrogels to identify the component responsible for the effect): inhibition of reactive oxygen species production by human polymorphonuclear neutrophils (PMNs) and in a cell-free system, oxygen consumption assay of PMNs (prior to oxidative burst), inhibition of human complement (limiting the generation of complement factors), mast cell degranulation, nitric oxide production by murine macrophages and TNF-alpha production by human monocytes/macrophages. Where toxicity could cause cell inhibition, cell viability was assessed. PVP-ILH and its components interacted in our series of bioassays at various stages in the inflammation cascade. Scavenging of superoxide anions was the most pronounced effect. Furthermore, povidone-iodine inhibited PMN production of reactive oxygen species (inhibition of oxygen consumption) and a mast cell inhibitory (stabilising) activity was observed. Based on these results, the clinically observed, beneficial wound healing effects of PVP-ILH may also be attributed to an impediment of inflammatory activity, mainly by iodine's free radical scavenging. Controlling oxidative stress in the wound may be of great importance, especially since further reactions as, e.g., the formation of peroxynitrite from NO and ROS are prevented.

Automated live cell screening system based on a 24-well-microplate with integrated micro fluidics.

In research, pharmacologic drug-screening and medical diagnostics, the trend towards the utilization of functional assays using living cells is persisting. Research groups working with living cells are confronted with the problem, that common endpoint measurement methods are not able to map dynamic changes. With consideration of time as a further dimension, the dynamic and networked molecular processes of cells in culture can be monitored. These processes can be investigated by measuring several extracellular parameters. This paper describes a high-content system that provides real-time monitoring data of cell parameters (metabolic and morphological alterations), e.g., upon treatment with drug compounds. Accessible are acidification rates, the oxygen consumption and changes in adhesion forces within 24 cell cultures in parallel. Addressing the rising interest in biomedical and pharmacological high-content screening assays, a concept has been developed, which integrates multi-parametric sensor readout, automated imaging and probe handling into a single embedded platform. A life-maintenance system keeps important environmental parameters (gas, humidity, sterility, temperature) constant.

Chemotherapy of brain tumour using doxorubicin bound to surfactant-coated poly(butyl cyanoacrylate) nanoparticles: revisiting the role of surfactants.

Poly(butyl cyanoacrylate) nanoparticles coated with poloxamer 188 (Pluronic) F68) and also, as shown previously, polysorbate 80 (Tween 80) considerably enhance the anti-tumour effect of doxorubicin against an intracranial glioblastoma in rats. The investigation of plasma protein adsorption on the surface of the drug-loaded nanoparticles by two-dimensional electrophoresis (2-D PAGE) revealed that both surfactants, besides other plasma components, induced a considerable adsorption of apolipoprotein A-I (ApoA-I). It is hypothesized that delivery of doxorubicin to the brain by means of nanoparticles may be augmented by the interaction of apolipoprotein A-I that is anchored on the surface of the nanoparticles with the scavenger receptor class B type I (SR-BI) located at the blood-brain barrier. This is the first study that shows a correlation between the adsorption of apolipoprotein A-I on the nanoparticle surface and the delivery of the drug across the blood-brain barrier.

A ratiometric imaging method for mapping ion flux densities.

A heterogeneous distribution of ion channels on the cell surface is a prerequisite for several cellular functions. Thus, there has been considerable interest in methods allowing the mapping of ion channel distributions. Here we report on a novel ratiometric imaging technique appropriate to measure spatially resolved ion flux signals by using ion sensitive dyes. However, given that certain relevant cell properties like the surface to volume ratio may exhibit significant spatial heterogeneities, the local influx signal cannot be interpreted as a measure of the local open channel concentration or flux density. To overcome this problem, we suggest an internal normalization procedure, which, in analogy to, but clearly distinct from, well-established ratioing techniques, eliminates effects which would otherwise obscure the desired result. Ratioing is performed on flux signals from a given cell, triggered by two different, subsequent stimuli. If the two stimuli address different ion channels, the flux density distribution caused by two channel types can be determined relative to each other. In cases where one of the stimuli triggers a spatially homogeneous flux signal, ratioing yields an ion flux density map for a given channel type. Thus distribution patterns of ion channels active during a given stimulus may be derived.

Bus, a bushy Arabidopsis CYP79F1 knockout mutant with abolished synthesis of short-chain aliphatic glucosinolates.

A new mutant of Arabidopsis designated bus1-1 (for bushy), which exhibited a bushy phenotype with crinkled leaves and retarded vascularization, was characterized. The phenotype was caused by an En-1 insertion in the gene CYP79F1. The deduced protein belongs to the cytochrome P450 superfamily. Because members of the CYP79 subfamily are believed to catalyze the oxidation of amino acids to aldoximes, the initial step in glucosinolate biosynthesis, we analyzed the level of glucosinolates in a CYP79F1 null mutant (bus1-1f) and in an overexpressing plant. Short-chain glucosinolates derived from methionine were completely lacking in the null mutant and showed increased levels in the overexpressing plant, indicating that CYP79F1 uses short-chain methionine derivatives as substrates. In addition, the concentrations of indole-3-ylmethyl-glucosinolate and the content of the auxin indole-3-acetic acid and its precursor indole-3-acetonitrile were increased in the bus1-1f mutant. Our results demonstrate for the first time that the formation of glucosinolates derived from methionine is mediated by CYP79F1 and that knocking out this cytochrome P450 has profound effects on plant growth and development.

A bench-top method for evaluating modular total hip component combinations.

A modular total hip prosthesis allows the surgeon to vary head size, neck length, and acetabular coverage. Different combinations, however, may increase the risk of impingement of the prosthesis neck on the acetabular liner, leading to dislocation. We developed a bench-top model to test different modular component combinations to determine how far the femoral component could travel within the acetabular liner before impingement led to dislocation. We tested two hip systems, each from a different manufacturer. Certain components increased the risk of impingement and dislocation: skirted necks, smaller heads, and lipped liners. While the contribution of each component was small, the risk was additive, so that the combination of a small head with a skirted neck in a lipped liner actually reduced the available range of motion by 18 degrees, from 146 degrees to 128 degrees, in the first system, and by 13 degrees, from 156 degrees to 143 degrees in the other. This method could be adapted to test a wide range of component configurations, from a number of manufacturers. These findings may be useful in avoiding potentially unstable configurations when given a choice of modular components.

Laser atomic absorption spectrometry of excited Hg in a discharge applying sum frequency mixing of two diode lasers (preliminary results).

Wavelength modulation diode laser atomic absorption spectrometry is applied to the detection of atomic mercury. Transitions from metastable energy levels highly populated in a radio-frequency discharge are induced with laser diodes by use of nonlinear techniques. The wavelength of one strong transition at 365.119 nm with a high oscillator strength is obtained by sum frequency generation of two diode lasers. The cold vapor technique is used to transfer ionic into atomic mercury. The mercury in the vapor phase is transported by an argon stream into the discharge tube. From the time-dependent absorption signals detection limits of 100 ng/L are achieved at this state of research.

Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage.

The signal and limitations of calcium florescence imaging using nonresonant multiphoton absorption of near-infrared femto- and picosecond laser pulses were examined. The fluorescence changes of various Ca(2+)-indicators induced by transient increases of the intradendritic calcium concentration were evaluated by evoking physiological activity in neocortical neurons in rat brain slices. Photodamage was noticeable as irreversible changes in the parameters describing the calcium fluorescence transients. At higher two-photon excitation rates, a great variety of irregular functional and structural alterations occurred. Thus, signal and observation time were limited by phototoxic effects. At lower excitation rates, photodamage accumulated linearly with exposure time. Femtosecond and picosecond laser pulses were directly compared with respect to this cumulative photodamage. The variation of the pulse length at a constant two-photon excitation rate indicated that a two-photon excitation mechanism is mainly responsible for the cumulative photodamage within the investigated window of 75 fs to 3.2 ps. As a direct consequence, at low excitation rates, the same image quality is achieved irrespective of whether two-photon Ca(2+)-imaging is carried out with femto- or picosecond laser pulses.

Control of phobic behavioral responses by rhodopsin-induced photocurrents in Chlamydomonas.

Both phototactic and photophobic responses of Chlamydomonas are mediated by a visual system comprising a rhodopsin photoreceptor. Suction pipette recordings have revealed that flash stimulation causes calcium currents into the eyespot and the flagella. These photocurrents have been suggested to be the trigger for all behavioral light responses of the cell. But this has never been shown experimentally. Here we describe a detection technique that combines electrical and optical measurements from individual algae held in a suction pipette. Thus it is possible to record photocurrents and flagellar beating simultaneously and establish a direct link between the two. We demonstrate that in Chlamydomonas only the photoreceptor current in conjuction with a fast flagellar current constitutes the trigger for photophobic responses. Within the time of the action-potential-like flagellar current, the flagella switch from forward to backward swimming, which constitutes the beginning of the photoshock reaction. The switch is accompanied by a complex frequency change and beating pattern modulation. The results are interpreted in terms of a general model for phototransduction in green algae (Chlorophyceae).

How Chlamydomonas keeps track of the light once it has reached the right phototactic orientation.

By using a real-time assay that allows measurement of the phototactic orientation of the unicellular alga Chlamydomonas with millisecond time resolution, it can be shown that single photons not only induce transient direction changes but that fluence rates as low as 1 photon cell(-1) s(-1) can already lead to a persistent orientation. Orientation is a binary variable, i.e., in a partially oriented population some organisms are fully oriented while the rest are still at random. Action spectra reveal that the response to a pulsed stimulus follows the Dartnall-nomogram for a rhodopsin while the response to a persistent stimulus falls off more rapidly toward the red end of the spectrum. Thus light of 540 nm, for which chlamy-rhodopsin is equally sensitive as for 440-nm light, induces no measurable persistent orientation while 440-nm light does. A model is presented which explains not only this behavior, but also how Chlamydomonas can track the light direction and switches between a positive and negative phototaxis. According to the model the ability to detect the direction of light, to make the right turn and to stay oriented, is a direct consequence of the helical path of the organism, the orientation of its eyespot relative to the helix-axis, and the special shielding properties of eyespot and cell body. The model places particular emphasis on the fact that prolonged swimming into the correct direction not only requires making a correct turn initially, but also avoiding further turns once the right direction has been reached.

A microspectrophotometric study of the shielding properties of eyespot and cell body in Chlamydomonas.

The eyespot apparatus of the unicellular alga Chlamydomonas exhibits a clear directivity, i.e., it perceives light from different directions with different sensitivity. Using a newly constructed confocal microscope we have studied how absorption and reflection of eyespot and cell body shape this directivity. In agreement with previous results the eyespot was found to be highly reflectant, owing to its interference reflector design, but only for yellow light. Light of 490 nm, the maximum of absorption of the photoreceptor, was hardly reflected at all, even when the reflector was "tuned" to lower wavelengths by tilting it relative to the incoming light. The absorption of the carotenoids in the interference reflector also contributed little to the shielding properties of the cell, leaving the major contribution to the cell body. Thus most of the attenuation of light reaching the eyespot from the rear is due to chlorophyll and other pigments within the cell. In its peak around 490 nm the "contrast-ratio" reached a value of 8-10.

Dorsal capsulodesis using suture anchors.

Scapholunate instability can be treated by stabilizing the scaphoid with the dorsal wrist capsule, which acts as a tether to control palmar rotation. We began using suture anchors instead of pull-out wires to secure the dorsal capsule to the scaphoid. Thirty-eight patients were treated with this method, and 35 were followed an average of 24 months. Sixteen were pain free, 12 had pain with activity, and 7 required additional surgical treatment. Patients regained 85% of expected grip strength, with 25 of the 35 returning to their preinjury job. The scapholunate gap, reduced at the time of surgery, recurred in 31 of the 35 patients, but this alone did not correlate with symptoms. This procedure improves symptoms in a majority of patients, but like other procedures described for scapholunate instability, it does not completely cure the problem.

[Prescribing clozapine to that patient: between anarchistic empiricism and arbitrary classification]. / Prescrire la clozapine à ce patient-là: entre l'empirisme anarchique et le codage arbitraire.

Opportunities for prescribing Leoponex are dependent on the notion of resistance schizophrenia as determined by rating scales. This does not do justice to the finer points of clinical assessment, which occasionally argue in favour of wider indications. The question of early treatment also arises in certain individual cases.

Instrumentation for multiwavelengths excitation imaging.

We have developed a fluorescence ratio-imaging system which is based on a 12 bit, 2 MHz slow scan CCD camera and a patented (patent number P 42 28 366.3-52) polychromatic illumination system. The latter produces monochromatic light (12 nm bandwidth) of high intensity (> 3 mW between 300 and 500 nm) and allows one to switch to any wavelength between 260 and 680 nm in less than 3.5 ms in a computer-controlled fashion. The possibility to execute complex wavelength protocols facilitates multiple dye measurements with optimal exposure time for a given wavelength and the return to a dark phase in between exposures. Moreover, it allows sweeping over extended spectral regions in order to determine optimal experimental conditions for a given task. Wavelength selection is performed by a diffraction grating which is mounted onto a galvanometric scanner. The grating is illuminated by white light from a 75 W xenon lamp, using exclusively reflective optics, and the diffracted monochromatic light is coupled into the microscope by means of a single fibre quartz light guide. The epifluorescence optics, a special, achromatic, aplanatic UV condensor, image the exit face plate of the fibre into the specimen plane of an inverted microscope. This 'critical illumination' yields better homogeneity in the specimen plane than the classical Köhler illumination. Thus, with the Zeiss Fluar objective 40 x, NA = 1.3, fluence rates close to 10(23) photons m2 s-1 may be achieved at 340 nm. A DOS programme has been written in 'C' which controls both the monochromator and slow scan imaging system. It can acquire up to 13 full frames per s, and in its binning and skipping mode up to 100 subframes per s may be acquired. The frame-transfer structure of the chip allows one to acquire images at wavelength 'B' while simultaneously writing image data previously acquired at wavelength 'A' into the computer.

Progressive scapulothoracic subluxation after fracture of the clavicle.

Fracture of the clavicle is a common injury that usually heals without consequence. Instability of the scapulothoracic articulation does not usually occur. In this case, the scapulothoracic subluxation was not obvious at the time of injury, but progressively worsened over the following 2 weeks. Open reduction and internal fixation of the clavicle fracture restored stability to the shoulder girdle. The patient went on to regain full function of her injured extremity.