Use of a short hPDE6b promoter for rod gene transfer in a model of severe retinal cystrophy, the Rd10 mouse

Purpose: Gene therapy of severe retinal dystrophies directly affecting photoreceptor is still a challenge in terms of clinical application. One of the main hurdles is to generate high transgene expression specifically in rods or cones. In the present study, we are investigating the possibility to drive hPDE6b expression in the Rd10 mouse retina using a specific sequence of the human PDE6b promoter. Methods: Two 5' flanking fragments of the human PDE6b gene: (-93 to +53 (146 bp) and -297 to +53 (350 bp, see Di Polo and Farber, 1995) were cloned in different plasmids in order to check their expression in vitro and in vivo. These elements drove the activity of either luciferase (pGL3 plasmids) or EGFP (AAV2/8 backbone). Then, an AAV2/8 vector carrying the PDE6b cDNA was tested with subretinal injections at P9 in the Rd10 eyes. Eye fundus, OCT, ERG recordings and histological investigations were performed to assess the efficacy of the gene transfer. Results: The short PDE6b promoter containing 146bp (-93 to +53) showed the highest activity in the Y-79 cells, as described previously (Di Polo and Farber, 1995). Subretinal administrations of AAV2/8-PDE6bpromoter-EGFP allowed a rapid expression specifically in rods and not in cones. The expression is faster than a vector containing the CMV promoter. The AAV2/8-PDE6bpromoter-PDE6b and the control vector were injected at P9 in the Rd10 mouse retina and investigated 5 weeks post-injection. Out of 14 eyes, 6 presented an increased rod sensitivity of about 300 fold, and increased a- and b-wave responses in ERG recordings. Flicker stimulations revealed that cones are also functional. OCT images and histological analyses revealed an increased ONL size in the injected area. The retina treated with the therapeutic vector presented 4-6 rows of photoreceptors with outersegments containing PDE6b. In the control eyes, only 2-4 rows of photoreceptors with almost no OS were observed . Conclusions: The 146 bp promoter sequence (-93 to + 53) is the shortest regulatory element described to date which allows to obtain efficient rod-specific expression in the context of somatic gene transfer. This first result is of great interest for AAV vector design in general allowing more space for the accommodation of transgenes of interest and good expression in rods. Moreover we showed the proof of principle of the efficacy of AAV2/8-PDE6bp-PDE6b vector in the Rd10 mouse model of severe photoreceptor degeneration without using neither AAV mutated capsids, nor self-complementary vectors.

Young infants' triangular communication with their parents in the context of maternal postpartum psychosis: Four case studies

With increasing data on the dynamics of normative couples as they transition to parenthood and become a triad, the need for greater understanding of the impact of parental psychopathology on this transition has become clear. The goal of the current article is to begin exploring this area that has received little attention to date, by describing case examples from a study of clinical families as they transitioned to parenthood. Four representative cases were selected from a pool of 13 mother-father-baby triads, for whom the mother had been hospitalized conjointly with her infant due to a psychotic episode during the postpartum period. The families were observed as part of a clinical consultation that included a semistructured play paradigm known as the Lausanne Trilogue Play (LTP; E. Fivaz-Depeursinge, & A. Corboz-Warnery, 1999). Interactions were scored using standardized measures as well as clinical impressions. All families from the clinical sample were noted to struggle and frequently failed to achieve the goals of play. The impact on the infants in terms of their developing sense of self as well as their defensive strategies in this context are discussed, with clinical implications explored.

Considering context in psychological leadership research

Despite Lewin's identification of the importance of context in behavioral research over 70 years ago, leadership psychology tended to ignore the context. Only in the past 10 years has the context been more routinely included in psychological leadership research. We provide examples of leadership research that has explored the context, introduce the special issue articles, and provide suggestions for future research on the context of leadership.

Symptoms, comorbidity, and clinical course of depression in immigrants: Putting psychopathology in context.

BACKGROUND: Migration is considered a depression risk factor when associated with psychosocial adversity, but its impact on depression's clinical characteristics has not been specifically studied. We compared 85 migrants to 34 controls, examining depression's severity, symptomatology, comorbidity profile and clinical course. METHOD: A MINI interview modified to assess course characteristics was used to assign DSM-IV axis I diagnoses; medical files were used for Somatoform Disorders. Severity was assessed with the Montgomery-Asberg scale. Wherever possible, we adjusted comparisons for age and gender using logistic and linear regressions. RESULTS: Depression in migrants was characterized by higher comorbidity (mostly somatoform and anxiety disorders), higher severity, and a non-recurrent, chronic course. LIMITATIONS: Our sample comes from a single center, and should be replicated in other health care facilities and other countries. Somatoform disorder diagnoses were solely based on file-content. CONCLUSION: Depression in migrants presented as a complex, chronic clinical picture. Most of our migrant patients experienced significant psychosocial adversity before and after migration: beyond cultural issues, our results suggest that psychosocial adversity impacts on the clinical expression of depression. Our study also suggests that migration associated with psychosocial adversity might play a specific etiological role, resulting in a distinct clinical picture, questioning the DSM-IV unitarian model of depression. The chronic course might indicate a resistance to standard therapeutic regimen and hints at the necessity of developing specific treatment strategies, adapted to the individual patients and their specific context.

Tissue biomarker development in a multicentre trial context: a feasibility study on the PETACC3 stage II and III colon cancer adjuvant treatment trial.

PURPOSE: We evaluated the feasibility of biomarker development in the context of multicenter clinical trials. EXPERIMENTAL DESIGN: Formalin-fixed, paraffin-embedded (FFPE) tissue samples were collected from a prospective adjuvant colon cancer trial (PETACC3). DNA was isolated from tumor as well as normal tissue and used for analysis of microsatellite instability, KRAS and BRAF genotyping, UGT1A1 genotyping, and loss of heterozygosity of 18 q loci. Immunohistochemistry was used to test expression of TERT, SMAD4, p53, and TYMS. Messenger RNA was retrieved and tested for use in expression profiling experiments. RESULTS: Of the 3,278 patients entered in the study, FFPE blocks were obtained from 1,564 patients coming from 368 different centers in 31 countries. In over 95% of the samples, genomic DNA tests yielded a reliable result. Of the immmunohistochemical tests, p53 and SMAD4 staining did best with reliable results in over 85% of the cases. TERT was the most problematic test with 46% of failures, mostly due to insufficient tissue processing quality. Good quality mRNA was obtained, usable in expression profiling experiments. CONCLUSIONS: Prospective clinical trials can be used as framework for biomarker development using routinely processed FFPE tissues. Our results support the notion that as a rule, translational studies based on FFPE should be included in prospective clinical trials.

MRSA screening by the Xpert MRSA PCR assay: pooling samples of the nose, throat, and groin increases the sensitivity of detection without increasing the laboratory costs.

The performance of the Xpert MRSA polymerase chain reaction (PCR) assay on pooled nose, groin, and throat swabs (three nylon flocked eSwabs into one tube) was compared to culture by analyzing 5,546 samples. The sensitivity [0.78, 95 % confidence interval (CI) 0.73-0.82] and specificity (0.99, 95 % CI 0.98-0.99) were similar to the results from published studies on separated nose or other specimens. Thus, the performance of the Xpert MRSA assay was not affected by pooling the three specimens into one assay, allowing a higher detection rate without increasing laboratory costs, as compared to nose samples alone.

Enhanced sensitivity detection of protein immobilization by fluorescent interference on oxidized silicon.

We present a silicon chip-based approach for the enhanced sensitivity detection of surface-immobilized fluorescent molecules. Green fluorescent protein (GFP) is bound to the silicon substrate by a disuccinimidyl terephtalate-aminosilane immobilization procedure. The immobilized organic layers are characterized by surface analysis techniques, like ellipsometry, atomic force microscopy (AFM) and X-ray induced photoelectron spectroscopy. We obtain a 20-fold enhancement of the fluorescent signal, using constructive interference effects in a fused silica dielectric layer, deposited before immobilization onto the silicon. Our method opens perspectives to increase by an order of magnitude the fluorescent response of surface immobilized DNA- or protein-based layers for a variety of biosensor applications.

Increased mobility of major histocompatibility complex I-peptide complexes decreases the sensitivity of antigen recognition.

CD8(+) cytotoxic T lymphocytes (CTL) can recognize and kill target cells expressing only a few cognate major histocompatibility complex (MHC) I-peptide complexes. This high sensitivity requires efficient scanning of a vast number of highly diverse MHC I-peptide complexes by the T cell receptor in the contact site of transient conjugates formed mainly by nonspecific interactions of ICAM-1 and LFA-1. Tracking of single H-2K(d) molecules loaded with fluorescent peptides on target cells and nascent conjugates with CTL showed dynamic transitions between states of free diffusion and immobility. The immobilizations were explained by association of MHC I-peptide complexes with ICAM-1 and strongly increased their local concentration in cell adhesion sites and hence their scanning by T cell receptor. In nascent immunological synapses cognate complexes became immobile, whereas noncognate ones diffused out again. Interfering with this mobility modulation-based concentration and sorting of MHC I-peptide complexes strongly impaired the sensitivity of antigen recognition by CTL, demonstrating that it constitutes a new basic aspect of antigen presentation by MHC I molecules.

Improving the sensitivity of the sequence profile method.

The sequence profile method (Gribskov M, McLachlan AD, Eisenberg D, 1987, Proc Natl Acad Sci USA 84:4355-4358) is a powerful tool to detect distant relationships between amino acid sequences. A profile is a table of position-specific scores and gap penalties, providing a generalized description of a protein motif, which can be used for sequence alignments and database searches instead of an individual sequence. A sequence profile is derived from a multiple sequence alignment. We have found 2 ways to improve the sensitivity of sequence profiles: (1) Sequence weights: Usage of individual weights for each sequence avoids bias toward closely related sequences. These weights are automatically assigned based on the distance of the sequences using a published procedure (Sibbald PR, Argos P, 1990, J Mol Biol 216:813-818). (2) Amino acid substitution table: In addition to the alignment, the construction of a profile also needs an amino acid substitution table. We have found that in some cases a new table, the BLOSUM45 table (Henikoff S, Henikoff JG, 1992, Proc Natl Acad Sci USA 89:10915-10919), is more sensitive than the original Dayhoff table or the modified Dayhoff table used in the current implementation. Profiles derived by the improved method are more sensitive and selective in a number of cases where previous methods have failed to completely separate true members from false positives.

Integration of high-resolution geophysical data for the characterization of alluvial aquifers : new approaches and implications for hydrological predictions

Abstract Accurate characterization of the spatial distribution of hydrological properties in heterogeneous aquifers at a range of scales is a key prerequisite for reliable modeling of subsurface contaminant transport, and is essential for designing effective and cost-efficient groundwater management and remediation strategies. To this end, high-resolution geophysical methods have shown significant potential to bridge a critical gap in subsurface resolution and coverage between traditional hydrological measurement techniques such as borehole log/core analyses and tracer or pumping tests. An important and still largely unresolved issue, however, is how to best quantitatively integrate geophysical data into a characterization study in order to estimate the spatial distribution of one or more pertinent hydrological parameters, thus improving hydrological predictions. Recognizing the importance of this issue, the aim of the research presented in this thesis was to first develop a strategy for the assimilation of several types of hydrogeophysical data having varying degrees of resolution, subsurface coverage, and sensitivity to the hydrologic parameter of interest. In this regard a novel simulated annealing (SA)-based conditional simulation approach was developed and then tested in its ability to generate realizations of porosity given crosshole ground-penetrating radar (GPR) and neutron porosity log data. This was done successfully for both synthetic and field data sets. A subsequent issue that needed to be addressed involved assessing the potential benefits and implications of the resulting porosity realizations in terms of groundwater flow and contaminant transport. This was investigated synthetically assuming first that the relationship between porosity and hydraulic conductivity was well-defined. Then, the relationship was itself investigated in the context of a calibration procedure using hypothetical tracer test data. Essentially, the relationship best predicting the observed tracer test measurements was determined given the geophysically derived porosity structure. Both of these investigations showed that the SA-based approach, in general, allows much more reliable hydrological predictions than other more elementary techniques considered. Further, the developed calibration procedure was seen to be very effective, even at the scale of tomographic resolution, for predictions of transport. This also held true at locations within the aquifer where only geophysical data were available. This is significant because the acquisition of hydrological tracer test measurements is clearly more complicated and expensive than the acquisition of geophysical measurements. Although the above methodologies were tested using porosity logs and GPR data, the findings are expected to remain valid for a large number of pertinent combinations of geophysical and borehole log data of comparable resolution and sensitivity to the hydrological target parameter. Moreover, the obtained results allow us to have confidence for future developments in integration methodologies for geophysical and hydrological data to improve the 3-D estimation of hydrological properties.

Mineralogical, petrographic and geochemical characterisation of white and coloured Iberian marbles in the context of the provenancing of some artefacts from Thamusida (Kenitra, Morocco)

A multi-analytical study has been carried out on a collection of white and coloured Iberian marbles. A total of 135 marble specimens were collected in Spain and Portugal from the Betic chain (Alhaurin de la Torre, Mijas, Macael), Ossa Morena (Alconera, Almaden de la Plata and Viana do Alentejo), and the Estremoz Anticline (Bencatel, Borba and Estremoz) areas. X-ray diffractometry and carbon and oxygen stable isotope analysis were carried out on these samples; 38 samples were also investigated by optical and scanning electron microscopy. The results provide a set of diagnostic parameters that allow discriminating the sampled marble quarries. The carbonate minerals composition is distinctive for the Mijas and Alhaurin de la Torre marbles; the isotopic analysis allows discriminating also between these two dolomitic marble quarries. The Ossa Morena and Estremoz Anticline marbles share a similar stable isotope composition; the accessory mineral content, the maximum grain size (MGS) and the fabric are particularly useful in the distinction between them. In the framework of archaeometric provenance studies on Thamusida (Kenitra, Morocco) Roman marble artefacts, a specific comparison between this new Iberian database and archaeological findings has been carried out. The hypothesis of commercial exchanges between the Iberian regions and Roman Morocco is supported by the results of the provenance study, which suggested the Almaden de la Plata and Mijas quarries as possible sources of raw materials for the production of archaeological artefacts.