Iridocyclectomy for neovascular glaucoma caused by proton-beam radiotherapy of pigmented ciliary adenocarcinoma.

PURPOSE: To report neovascular glaucoma after proton-beam radiotherapy of an adenocarcinoma of the pigmented ciliary epithelium and its successful treatment by iridocyclectomy. PATIENT AND METHODS: A 65-year-old man developed neovascular glaucoma 10 months after proton-beam radiotherapy of a small pigmented iridociliary tumour with a clinical differential diagnosis of uveal melanoma or adenocarcinoma. The diagnosis of 'toxic tumour syndrome' was made, and iridocyclectomy performed. RESULTS: Histopathology and immunohistochemistry of the specimen diagnosed an adenocarcinoma of the pigmented ciliary epithelium, with the presence of mitoses suggesting residual viable tumour cells. The rubeosis regressed, with normalization of the intraocular pressure. Phacoemulsification for radiation-induced cataract restored VA to 6/9, which was better than that recorded at initial referral, the patient having longstanding cellophane maculopathy. CONCLUSIONS: Neovascular glaucoma after radiotherapy of a small, pigmented, ciliary body tumour raises the possibility of adenocarcinoma. This 'toxic tumour syndrome' may respond to iridocyclectomy of the irradiated tumour.

Laser scanning evaluation of atrophy after autologous free muscle transfer.

Denervated muscle tissue undergoes morphologic changes that result in atrophy. The amount of muscle atrophy after denervation following free muscle transfer has not been measured so far. Therefore, the amount of muscle atrophy in human free muscle transfer for lower extremity reconstruction was measured in a series of 10 patients. Three-dimensional laser surface scanning was used to measure flap volume changes 2 weeks as well as 6 and 12 months after the operation. None of the muscles transferred was re-innervated.All muscles healed uneventfully without signs of compromised perfusion resulting in partial flap loss. The muscle volume decreased to 30 ± 4% and 19 ± 4% 6 and 12 months, respectively, after the operation, ie, the volume decreased by approximately 80% within a 12-month period.Denervated free muscle flap tissue undergoes massive atrophy of approximately 80%, mostly within the first 6 months.

Proton NMR of (15)N-choline metabolites enhanced by dynamic nuclear polarization.

Chemical shifts of protons can report on metabolic transformations such as the conversion of choline to phosphocholine. To follow such processes in vivo, magnetization can be enhanced by dynamic nuclear polarization (DNP). We have hyperpolarized in this manner nitrogen-15 spins in (15)N-labeled choline up to 3.3% by irradiating the 94 GHz electron spin resonance of admixed TEMPO nitroxide radicals in a magnetic field of 3.35 T during ca. 3 h at 1.2 K. The sample was subsequently transferred to a high-resolution magnet, and the enhanced polarization was converted from (15)N to methyl- and methylene protons, using the small (2,3)J((1)H,(15)N) couplings in choline. The room-temperature lifetime of nitrogen polarization in choline, T(1)((15)N) approximately 200 s, could be considerably increased by partial deuteration of the molecule. This procedure enables studies of choline metabolites in vitro and in vivo using DNP-enhanced proton NMR.

Use of phycoerythrin and allophycocyanin for fluorescence resonance energy transfer analyzed by flow cytometry: advantages and limitations.

BACKGROUND: This study validates the use of phycoerythrin (PE) and allophycocyanin (APC) for fluorescence energy transfer (FRET) analyzed by flow cytometry. METHODS: FRET was detected when a pair of antibody conjugates directed against two noncompetitive epitopes on the same CD8alpha chain was used. FRET was also detected between antibody conjugate pairs specific for the two chains of the heterodimeric alpha (4)beta(1) integrin. Similarly, the association of T-cell receptor (TCR) with a soluble antigen ligand was detected by FRET when anti-TCR antibody and MHC class I/peptide complexes (<<tetramers>>) were used. RESULTS: FRET efficiency was always less than 10%, probably because of steric effects associated with the size and structure of PE and APC. Some suggestions are given to take into account this and other effects (e.g., donor and acceptor concentrations) for a better interpretation of FRET results obtained with this pair of fluorochromes. CONCLUSIONS: We conclude that FRET assays can be carried out easily with commercially available antibodies and flow cytometers to study arrays of multimolecular complexes.

A genomic island present along the bacterial chromosome of the Parachlamydiaceae UWE25, an obligate amoebal endosymbiont, encodes a potentially functional F-like conjugative DNA transfer system.

BACKGROUND: The genome of Protochlamydia amoebophila UWE25, a Parachlamydia-related endosymbiont of free-living amoebae, was recently published, providing the opportunity to search for genomic islands (GIs). RESULTS: On the residual cumulative G+C content curve, a G+C-rich 19-kb region was observed. This sequence is part of a 100-kb chromosome region, containing 100 highly co-oriented ORFs, flanked by two 17-bp direct repeats. Two identical gly-tRNA genes in tandem are present at the proximal end of this genetic element. Several mobility genes encoding transposases and bacteriophage-related proteins are located within this chromosome region. Thus, this region largely fulfills the criteria of GIs. The G+C content analysis shows that several modules compose this GI. Surprisingly, one of them encodes all genes essential for F-like conjugative DNA transfer (traF, traG, traH, traN, traU, traW, and trbC), involved in sex pilus retraction and mating pair stabilization, strongly suggesting that, similarly to the other F-like operons, the parachlamydial tra unit is devoted to DNA transfer. A close relatedness of this tra unit to F-like tra operons involved in conjugative transfer is confirmed by phylogenetic analyses performed on concatenated genes and gene order conservation. These analyses and that of gly-tRNA distribution in 140 GIs suggest a proteobacterial origin of the parachlamydial tra unit. CONCLUSIONS: A GI of the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system. This is the first hint of a putative conjugative system in chlamydiae. Conjugation most probably occurs within free-living amoebae, that may contain hundreds of Parachlamydia bacteria tightly packed in vacuoles. Such a conjugative system might be involved in DNA transfer between internalized bacteria. Since this system is absent from the sequenced genomes of Chlamydiaceae, we hypothesize that it was acquired after the divergence between Parachlamydiaceae and Chlamydiaceae, when the Parachlamydia-related symbiont was an intracellular bacteria. It suggests that this heterologous DNA was acquired from a phylogenetically-distant bacteria sharing an amoebal vacuole. Since Parachlamydiaceae are emerging agents of pneumonia, this GI might be involved in pathogenicity. In future, conjugative systems might be developed as genetic tools for Chlamydiales.

Efficient lentiviral gene transfer into corneal stroma cells using a femtosecond laser.

We investigated a new procedure for gene transfer into the stroma of pig cornea for the delivery of therapeutic factors. A delimited space was created at 110 mum depth with a LDV femtosecond laser in pig corneas, and a HIV1-derived lentiviral vector expressing green fluorescent protein (GFP) (LV-CMV-GFP) was injected into the pocket. Corneas were subsequently dissected and kept in culture as explants. After 5 days, histological analysis of the explants revealed that the corneal pockets had closed and that the gene transfer procedure was efficient over the whole pocket area. Almost all the keratocytes were transduced in this area. Vector diffusion at right angles to the pocket's plane encompasses four (endothelium side) to 10 (epithelium side) layers of keratocytes. After 21 days, the level of transduction was similar to the results obtained after 5 days. The femtosecond laser technique allows a reliable injection and diffusion of lentiviral vectors to efficiently transduce stromal cells in a delimited area. Showing the efficacy of this procedure in vivo could represent an important step toward treatment or prevention of recurrent angiogenesis of the corneal stroma.

Fate of the herbicide glyphosate and its metabolite AMPA in soils and their transfer to surface waters: A multi-scale approach in the Lavaux vineyards, western Switzerland

The use of herbicides in agriculture may lead to environmental problems, such as surface water pollution, with a potential risk for aquatic organisms. The herbicide glyphosate is the most used active ingredient in the world and in Switzerland. In the Lavaux vineyards it is nearly the only molecule applied. This work aimed at studying its fate in soils and its transfer to surface waters, using a multi-scale approach: from molecular (10-9 m) and microscopic scales (10-6 m), to macroscopic (m) and landscape ones (103 m). First of all, an analytical method was developed for the trace level quantification of this widely used herbicide and its main by-product, aminomethylphosphonic acid (AMPA). Due to their polar nature, their derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) was done prior to their concentration and purification by solid phase extraction. They were then analyzed by ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). The method was tested in different aqueous matrices with spiking tests and validated for the matrix effect correction in relevant environmental samples. Calibration curves established between 10 and 1000ng/l showed r2 values above 0.989, mean recoveries varied between 86 and 133% and limits of detection and quantification of the method were as low as 5 and 10ng/l respectively. At the parcel scale, two parcels of the Lavaux vineyard area, located near the Lutrive River at 6km to the east of Lausanne, were monitored to assess to which extent glyphosate and AMPA were retained in the soil or exported to surface waters. They were equipped at their bottom with porous ceramic cups and runoff collectors, which allowed retrieving water samples for the growing seasons 2010 and 2011. Results revealed that the mobility of glyphosate and AMPA in the unsaturated zone was likely driven by the precipitation regime and the soil characteristics, such as slope, porosity structure and layer permeability discrepancy. Elevated glyphosate and AMPA concentrations were measured at 60 and 80 cm depth at parcel bottoms, suggesting their infiltration in the upper parts of the parcels and the presence of preferential flow in the studied parcels. Indeed, the succession of rainy days induced the gradual saturation of the soil porosity, leading to rapid infiltration through macropores, as well as surface runoff formation. Furthermore, the presence of more impervious weathered marls at 100 cm depth induced throughflows, the importance of which for the lateral transport of the herbicide molecules was determined by the slope steepness. Important rainfall events (>10 mm/day) were clearly exporting molecules from the soil top layer, as indicated by important concentrations in runoff samples. A mass balance showed that total loss (10-20%) mainly occurred through surface runoff (96%) and, to a minor extent, by throughflows in soils (4%), with subsequent exfiltration to surface waters. Observations made in the Lutrive River revealed interesting details of glyphosate and AMPA dynamics in urbanized landscapes, such as the Lavaux vineyards. Indeed, besides their physical and chemical properties, herbicide dynamics at the catchment level strongly depend on application rates, precipitation regime, land use and also on the presence of drains or constructed channels. Elevated concentrations, up to 4970 ng/l, observed just after the application, confirmed the diffuse export of these compounds from the vineyard area by surface runoff during main rain events. From April to September 2011, a total load of 7.1 kg was calculated, with 85% coming from vineyards and minor urban sources and 15% from arable crops. Small vineyard surfaces could generate high concentrations of herbicides and contribute considerably to the total load calculated at the outlet, due to their steep slopes (~10%). The extrapolated total amount transferred yearly from the Lavaux vineyards to the Lake of Geneva was of 190kg. At the molecular scale, the possible involvement of dissolved organic matter (DOM) in glyphosate and copper transport was studied using UV/Vis fluorescence spectroscopy. Combined with parallel factor (PARAFAC) analysis, this technique allowed characterizing DOM of soil and surface water samples from the studied vineyard area. Glyphosate concentrations were linked to the fulvic-like spectroscopic signature of DOM in soil water samples, as well as to copper, suggesting the formation of ternary complexes. In surface water samples, its concentrations were also correlated to copper ones, but not in a significant way to the fulvic-like signature. Quenching experiments with standards confirmed field tendencies in the laboratory, with a stronger decrease in fluorescence intensity for fulvic-like fluorophore than for more aromatic ones. Lastly, based on maximum concentrations measured in the river, an environmental risk for these compounds was assessed, using laboratory tests and ecotoxicity data from the literature. In our case and with the methodology applied, the risk towards aquatic species was found negligible (RF<1).

Functional expression of PHO1 to the Golgi and trans-Golgi network and its role in export of inorganic phosphate.

Arabidopsis thaliana PHO1 is primarily expressed in the root vascular cylinder and is involved in the transfer of inorganic phosphate (Pi) from roots to shoots. To analyze the role of PHO1 in transport of Pi, we have generated transgenic plants expressing PHO1 in ectopic A. thaliana tissues using an estradiol-inducible promoter. Leaves treated with estradiol showed strong PHO1 expression, leading to detectable accumulation of PHO1 protein. Estradiol-mediated induction of PHO1 in leaves from soil-grown plants, in leaves and roots of plants grown in liquid culture, or in leaf mesophyll protoplasts, was all accompanied by the specific release of Pi to the extracellular medium as early as 2-3 h after addition of estradiol. Net Pi export triggered by PHO1 induction was enhanced by high extracellular Pi and weakly inhibited by the proton-ionophore carbonyl cyanide m-chlorophenylhydrazone. Expression of a PHO1-GFP construct complementing the pho1 mutant revealed GFP expression in punctate structures in the pericycle cells but no fluorescence at the plasma membrane. When expressed in onion epidermal cells or in tobacco mesophyll cells, PHO1-GFP was associated with similar punctate structures that co-localized with the Golgi/trans-Golgi network and uncharacterized vesicles. However, PHO1-GFP could be partially relocated to the plasma membrane in leaves infiltrated with a high-phosphate solution. Together, these results show that PHO1 can trigger Pi export in ectopic plant cells, strongly indicating that PHO1 is itself a Pi exporter. Interestingly, PHO1-mediated Pi export was associated with its localization to the Golgi and trans-Golgi networks, revealing a role for these organelles in Pi transport.

Genomic islands: tools of bacterial horizontal gene transfer and evolution.

Abstract Bacterial genomes evolve through mutations, rearrangements or horizontal gene transfer. Besides the core genes encoding essential metabolic functions, bacterial genomes also harbour a number of accessory genes acquired by horizontal gene transfer that might be beneficial under certain environmental conditions. The horizontal gene transfer contributes to the diversification and adaptation of microorganisms, thus having an impact on the genome plasticity. A significant part of the horizontal gene transfer is or has been facilitated by genomic islands (GEIs). GEIs are discrete DNA segments, some of which are mobile and others which are not, or are no longer mobile, which differ among closely related strains. A number of GEIs are capable of integration into the chromosome of the host, excision, and transfer to a new host by transformation, conjugation or transduction. GEIs play a crucial role in the evolution of a broad spectrum of bacteria as they are involved in the dissemination of variable genes, including antibiotic resistance and virulence genes leading to generation of hospital 'superbugs', as well as catabolic genes leading to formation of new metabolic pathways. Depending on the composition of gene modules, the same type of GEIs can promote survival of pathogenic as well as environmental bacteria.

Lentiviral vector-mediated gene transfer in adult mouse photoreceptors is impaired by the presence of a physical barrier

RESUME L'utilisation de la thérapie génique dans l'approche d'un traitement des maladies oculaires dégénératives, plus particulièrement de la rétinite pigmentaire, semble être très prometteuse (Acland et al. 2001). Parmi les vecteurs développés, les vecteurs lentiviraux (dérivé du virus humain HIV-1), permettent la transduction des photorécepteurs après injection sous-rétinienne chez la souris durant les premiers jours de vie. Cependant l'efficacité du transfert de gène est nettement plus limitée dans ce type cellulaire après injection chez l'adulte (Kostic et al. 2003). L'objet de notre étude est de déterminer si la présence d'une barrière physique produite au cours du développement, située entre les photorécepteurs et l'épithélium pigmentaire ainsi qu'entre les photorécepteurs eux-mêmes, est responsable de: la diminution de l'entrée en masse du virus dans les photorécepteurs, minimisant ainsi son efficacité chez la souris adulte. De précédentes recherches, chez le lapin, ont décrit la capacité d'enzymes spécifiques comme la Chondroïtinase ABC et la Neuraminidase X de modifier la structure de la matrice entourant les photorécepteurs (Inter Photoreceptor Matrix, IPM) par digestion de certains de ses constituants suite à leur injection dans l'espace sous-rétinien (Yao et al. 1990). Considérant l'IPM comme une barrière physique, capable de réduire l'efficacité de transduction des photorécepteurs chez la souris adulte, nous avons associé différentes enzymes simultanément à l'injection sous-rétinienne de vecteurs lentiviraux afin d'améliorer la transduction virale en fragilisant I'IPM, la rendant ainsi plus perméable à la diffusion du virus. L'injection sous-rétinienne de Neuraminidase X et de Chondroïtinase ABC chez la souris induit des modifications structurales de l'IPM qui se manifestent respectivement par la révélation ou la disparition de sites de liaison de la peanut agglutinin sur les photorécepteurs. L'injection simultanée de Neuraminidase X avec le vecteur viral contenant le transgène thérapeutique augmente significativement le nombre de photorécepteurs transduits (environ cinq fois). Nous avons en fait démontré que le traitement enzymatique augmente principalement la diffusion du lentivirus dans l'espace situé entre l'épithélium pigmentaire et les photorécepteurs. Le traitement à la Chondroïtinase ABC n'entraîne quant à elle qu'une légère amélioration non significative de la transduction. Cette étude montre qu'une meilleure connaissance de l'IPM ainsi que des substances capables de la modifier (enzymes, drogues etc.) pourrait aider à élaborer de nouvelles stratégies afin d'améliorer la distribution de vecteurs viraux dans la rétine adulte.

Proton Therapy for Uveal Melanoma in 43 Juvenile Patients: Long-Term Results.

PURPOSE: To examine the metastatic and survival rates, eye retention probability, and the visual outcomes of juvenile patients after proton beam radiotherapy (PBRT) for uveal melanoma (UM). DESIGN: Retrospective case-factor matched control study. PARTICIPANTS AND CONTROLS: Forty-three patients younger than 21 years treated with PBRT for UM were compared with 129 matched adult control patients. METHODS: Information on patient demographics and clinical characteristics were recorded before and after treatment from patients' files. The control group was composed of adult patients (>21 years) matched for tumor size (largest tumor diameter, ±2 mm; height, ±2 mm) and anterior margin location (iris, ciliary body, pre-equatorial or postequatorial choroid). For each juvenile patient, 3 adults were selected. MAIN OUTCOME MEASURES: Comparing outcomes of juvenile and adult patients in terms of metastatic and eye retention rates using the log-rank statistic, relative survival using the Hakulinen method, as well as their visual outcomes. RESULTS: Forty-three juvenile and 129 control cases were reviewed. The metastatic rate at 10 years was significantly lower in juvenile UM patients than in adult controls (11% vs. 34%; P <0.01), with an associated relative survival rate of 93% versus 65% (P = 0.02). Six juvenile patients (14%) demonstrated metastases. One patient underwent enucleation because of a presumed local tumor recurrence and 4 additional patients underwent enucleation because of complications (9.3%). In the adult control group, 27% (n = 35) of matched patients demonstrated metastases, there were 2 cases of local recurrence, and 16% (n = 21) underwent enucleation because of complications. A visual acuity of more than 0.10 was maintained in most cases, without any significant differences before or after treatment observed between both groups. CONCLUSIONS: After PBRT, metastatic and survival rates are significantly better for juvenile than for adult patients with UM. Clinically, juvenile and adult eyes react similarly to PBRT, with patients having a comparable eye retention probability and maintaining a useful level of vision in most cases. This is the largest case-control study of proton therapy in juvenile eyes to date and further validates PBRT as an appropriate conservative treatment for UM in patients younger than 21 years.

Bayesian Networks and the Value of the Evidence for the Forensic Two-Trace Transfer Problem

Forensic scientists face increasingly complex inference problems for evaluating likelihood ratios (LRs) for an appropriate pair of propositions. Up to now, scientists and statisticians have derived LR formulae using an algebraic approach. However, this approach reaches its limits when addressing cases with an increasing number of variables and dependence relationships between these variables. In this study, we suggest using a graphical approach, based on the construction of Bayesian networks (BNs). We first construct a BN that captures the problem, and then deduce the expression for calculating the LR from this model to compare it with existing LR formulae. We illustrate this idea by applying it to the evaluation of an activity level LR in the context of the two-trace transfer problem. Our approach allows us to relax assumptions made in previous LR developments, produce a new LR formula for the two-trace transfer problem and generalize this scenario to n traces.

Rpe65-Gene Transfer Using an Integration-Deficient Lentiviral Vector

Purpose: We previously demonstrated efficient retinal rescue of RPE65 mouse models (Rpe65-/- (Bemelmans et al, 2006) and Rpe65R91W/R91W mice) using a HIV1-derived lentiviral vector encoding for the mouse RPE65 cDNA. In order to optimize a lentiviral vector as an alternative tool for RPE65-derived Leber Congenital Amaurosis clinical trials, we evaluated the efficiency of an integration-deficient lentiviral vector (IDLV) encoding the human RPE65 cDNA to restore retinal function in the Rpe65R91W/R91W mice. Methods: An HIV-1-derived lentiviral vector expressing either the hrGFPII or the human Rpe65 cDNA under the control of a 0.8 kb fragment of the human Rpe65 promoter (R0.8) was produced by transient transfection of 293T cells. A LQ-integrase mutant was used to generate the IDLV vectors. IDLV-R0.8-hRPE65 or hrGFPII were injected subretinally into 1 month-old Rpe65R91W/R91W mice. Functional rescue was assessed by ERG (1 and 3 months post-injection) and cone survival by immunohistology. Results: An increased light sensitivity was detected by scotopic ERG in animals injected with IDLV-R0.8-hRPE65 compared to hrGFPII-treated animals or untreated mice. However the improvement was delayed compared to integration-proficient LV and observed at 3 months but not 1 month post-injection. Immunolabelling of cone markers showed an increased number of cones in the transduced area compared to control groups. Conclusions: The IDLV-R0.8-hRPE65 vectors allow retinal improvement in the Rpe65R91W/R91W mice. Both rod function and cone survival were demonstrated even if there is a delay in the rescue as assessed by scotopic ERG. Integration-deficient vectors minimize insertional mutagenesis and thus are safer candidates for human application. Further experiments using large animals are now needed to validate correct gene transfer and expression of the RPE65 gene as well as tolerance of the vector after subretinal injection before envisaging a clinical trial application.