Effect of retinol dehydrogenase gene transfer in a novel rat model of Stargardt disease.

Dysfunction of the ATPase-binding Cassette Transporter protein (ABCA4) can lead to early onset macular degeneration, in particular to Stargardt disease. To enable translational research into this form of blindness, we evaluated the effect of Cas9-induced disruptions of the ABCA4 gene to potentially generate new transgenic rat models of the disease. We show that deletion of the short exon preceding the second nucleotide-binding domain is sufficient to drastically knock down protein levels and results in accumulation of retinoid dimers similar to that associated with Stargardt disease. Overexpression of the retinol dehydrogenase enzymes RDH8 and RDH12 can to a limited extent offset the increase in the bisretinoid levels in the Abca4Ex42-/ - KO rats possibly by restricting the time window in which retinal can dimerize before being reduced to retinol. However, in vivo imaging shows that overexpression of RDH8 can induce retinal degeneration. This may be due to the depletion in the outer segment of the cofactor NADPH, needed for RDH function. The translational potential of RDH therapy as well as other Stargardt disease therapies can be tested using the Abca4 knockdown rat model.

Towards diversity in science - a glance at gender disparity in the Brazilian Society of Neuroscience and Behavior (SBNeC).

Gender equity is far from being achieved in most academic institutions worldwide. Women representation in scientific leadership faces multiple obstacles. Implicit bias and stereotype threat are considered important driving forces concerning gender disparities. Negative cultural stereotypes of weak scientific performance, unrelated to true capacity, are implicitly associated with women and other social groups, influencing, without awareness, attitudes and judgments towards them. Meetings of scientific societies are the forum in which members from all stages of scientific careers are brought together. Visibility in the scientific community stems partly from presenting research as a speaker. Here, we investigated gender disparities in the Brazilian Society of Neuroscience and Behavior (SBNeC). Across the 15 mandates (1978-2020), women occupied 30% of the directory board posts, and only twice was a woman president. We evaluated six meetings held between 2010 and 2019. During this period, the membership of women outnumbered that of men in all categories. A total of 57.50% of faculty members, representing the potential pool of speakers and chairs, were female. Compared to this expected value, female speakers across the six meetings were scarce in full conferences (χ2(5)=173.54, P<0.001) and low in symposia (χ2(5)=36.92, P<0.001). Additionally, women chaired fewer symposia (χ2(5)=47.83, P<0.001). Furthermore, men-chaired symposia had significantly fewer women speakers than women-chaired symposia (χ2(1)=56.44, P<0.001). The gender disparities observed here are similar to those in other scientific societies worldwide, urging them to lead actions to pursue gender balance and diversity. Diversity leads not only to fairness but also to higher-quality science.

Sensitivity of field isolates of Eimeria acervulina and E. maxima from three regions in Brazil to eight anticoccidial drugs.

Rotation with different active ingredients is among the most effective and recommended strategies to preserve the efficacy of anticoccidial drugs and reduce the emergence of resistance. Tools such as anticoccidial sensitivity tests (ASTs) are ideally used to make rational rotation programs and bring benefits to production. The objective of this study was to evaluate the sensitivity of E. acervulina (EA) and E. maxima (EM) from 3 different regions in Brazil, by using four ASTs. Feces samples weighing 6 to 7 kg were collected in the regions of São Paulo, Paraná, and Minas Gerais. Prevalent oocysts from feces were filtered, identified, and quantified to conduct 2 ASTs with EA and 2 with EM. The same experimental design was used in every AST (4 replicates per treatment, with 6 birds each, for a total of 240 birds). Treatment groups were a nonchallenged and nonmedicated control group (T1), a challenged and nonmedicated control group (T2), and the other groups challenged and treated with the following compounds: lasalocid (90 ppm - T3), maduramycin (6 ppm - T4), decoquinate (30 ppm - T5), nicarbazin+semduramicin (66 ppm - T6), monensin (110 ppm - T7), salinomycin (66 ppm - T8), narasin+nicarbazin (100 ppm - T9), and nicarbazin (125 ppm - T10). At the end of each AST (20 d), the percent change (delta value) between the treated group (T3 to T10) and the control group (T2) was calculated for the following variables: body weight gain, feed conversion ratio, lesion score, and an indicator of percentage of optimal anticoccidial activity (POAA) that included T2. Different sensitivity levels of EA and EM isolates could be identified. As a whole, drugs from T5 and T3 groups showed higher delta values when compared to other compounds, whereas the lowest sensitivity levels of these isolates were observed in groups T4 and T7. Despite some limiting factors, ASTs can be a good tool for strategic selection of anticoccidial drugs in order to maintain efficacy and extend the lifespan of these molecules.

Survivin drives tumor-associated macrophage reprogramming: a novel mechanism with potential impact for obesity.

PURPOSE: Recent studies point to adipose-derived stem cells (ASCs) as a link between obesity and cancer. We aimed to determine whether survivin, which is highly secreted by ASCs from subjects with obesity, might drive a pro-tumoral phenotype in macrophages. METHODS: The effect of ASC conditioned medium on the macrophage phenotype was assessed by expression studies. Survivin intracellular localization and internalization were examined by subcellular fractionation and immunofluorescence, respectively. Loss- and gain-of-function studies were performed using adenoviral vectors, and gene expression patterns, migration and invasion capacities of cancer cells were examined. Heterotypic cultures of ASCs, macrophages and cancer cells were established to mimic the tumor microenvironment. Survivin-blocking experiments were used to determine the impact of survivin on both macrophages and cancer cells. Immunohistochemical analysis of survivin was performed in macrophages from ascitic fluids of cancer patients and healthy controls. RESULTS: We found that obese-derived ASCs induced a phenotypic switch in macrophages characterized by the expression of both pro- and anti-inflammatory markers. Macrophages were found to internalize extracellular survivin, generating hybrid macrophages with a tumor-associated phenotype that included secretion of survivin. Exogenous expression of survivin in macrophages generated a similar phenotype and enhanced the malignant characteristics of cancer cells by a mechanism dependent on survivin phosphorylation at threonine 34. Survivin secreted by both ASCs from subjects with obesity and tumor-associated macrophages synergistically boosted the malignancy of cancer cells. Importantly, survivin was mainly detected in ascites-associated macrophages from patients with a malignant diagnosis. CONCLUSION: Our data indicate that survivin may serve as a molecular link between obesity and cancer and as a novel marker for tumor-associated macrophages.

Microencapsulated and uncoated butyric acid as alternative additives to the regeneration of intestinal mucosa in broilers challenged with Eimeria spp.

1. The effect of microencapsulated and uncoated butyric acid as an alternative to antibiotics on performance, intestinal morphology and regeneration of intestinal mucosa was studied in birds experimentally infected with Eimeria spp. 1 to 42 d-old.2. A total of 1,320 male Cobb® broiler chicks were allocated to one of five treatments in a completely randomised design, comprising a negative control, uncoated butyric acid (UA), microencapsulated butyric acid (MA), combined U + M butyric acid and a positive control (antibiotic+anticoccidial) in six replications. At 16 d-old, the birds were inoculated orally with 0.5 ml of a solution containing an Eimeria spp. pool.3. At 21 d of age, the birds receiving butyric acid alone had higher body weight gain (BWG) and feed intake (FI) compared to those supplemented with the blend of acids. For the total rearing period, in all variables, the positive control performed best (P < 0.001).4. At 14 d of age, birds that received diets containing UA had a deeper crypt depth in the jejunum than those fed diets containing microencapsulated acid (P = 0.0194). At 21 d of age, the birds fed the acids had higher villi (P = 0.0058) in the duodenum, compared to the negative control group.5. Supplementation with microencapsulated acid contributed to the intestinal health and recovery of post-challenge birds, but did not result in improvements in performance.

Agaricus subrufescens and Pleurotus ostreatus mushrooms as alternative additives to antibiotics in diets for broilers challenged with Eimeria spp.

1. The effect of A. subrufescens and P. ostreatus mushrooms as an alternative to antibiotics (avilamycin or monensin sodium) on performance, intestinal morphometry, immunity, and biochemical profile of broilers challenged with Eimeria spp. was studied from 1 to 42 d old. A total of 900 male Cobb® broiler chicks were distributed, according to a completely randomised design, into five treatments with six replicates each.2. The treatments consisted of: negative control (NC) - basal diet (BD) with no anticoccidial or antibiotic (non-challenged birds); negative control challenged (NCC) - NC fed to Eimeria spp. challenged birds; BD with 0.2% A. subrufescens inclusion for challenged birds (As), BD with 0.2% P. ostreatus inclusion for challenged birds (Po); and a positive control - BD with anticoccidial and antibiotic inclusion for challenged birds (ATB).3. At 11 d.o., the birds were each inoculated orally with 1 ml solution containing 2 × 105 sporulated oocysts/ml Eimeria acervulina and 2 × 104 sporulated oocysts/ml E. maxima and E. tenella.4. Birds subjected to Eimeria spp. challenge up to 21 d of age had greater crypt depth, indicating that the presence of undesirable microorganisms had an effect on cell proliferation.5. At 21 d old, the birds receiving ATB had higher average weight gain (AWG), feed intake (AFI), and feed conversion ratio (FCR) compared to those fed diets supplemented with mushrooms (As or Po). For the total rearing period (42 days), the birds that received ATB had higher AWG and AFI (P < 0.001) compared to those that received As or Po diets. Feeding avilamycin did not affect (P = 0.0676) FCR compared to the As or Po diet groups.6. From the morphometric and blood analyses there were no differences between broilers fed ATB, Po or As diets in either rearing periods. However, Po and As supplementation lowered blood triglyceride levels. At 21d there was a difference (P < 0.05) for MCV and haemoglobin, in which the mushrooms were similar to the antibiotic. At 42 d, there was a difference (P < 0.05) in haematocrit, erythrocyte, MCV, H: L, protein and albumin variables, in which the use of mushrooms was similar to the positive control, demonstrating that both (mushrooms and antibiotics) promoted a certain improvement in the health of the chickens.7. A. subrufescens and P. ostreatus can be used in broiler diets without compromising intestinal or haematological status, however, these ingredients did not result in improvements in performance.

Towards diversity in science - a glance at gender disparity in the Brazilian Society of Neuroscience and Behavior (SBNeC)

Gender equity is far from being achieved in most academic institutions worldwide. Women representation in scientific leadership faces multiple obstacles. Implicit bias and stereotype threat are considered important driving forces concerning gender disparities. Negative cultural stereotypes of weak scientific performance, unrelated to true capacity, are implicitly associated with women and other social groups, influencing, without awareness, attitudes and judgments towards them. Meetings of scientific societies are the forum in which members from all stages of scientific careers are brought together. Visibility in the scientific community stems partly from presenting research as a speaker. Here, we investigated gender disparities in the Brazilian Society of Neuroscience and Behavior (SBNeC). Across the 15 mandates (1978-2020), women occupied 30% of the directory board posts, and only twice was a woman president. We evaluated six meetings held between 2010 and 2019. During this period, the membership of women outnumbered that of men in all categories. A total of 57.50% of faculty members, representing the potential pool of speakers and chairs, were female. Compared to this expected value, female speakers across the six meetings were scarce in full conferences (χ2(5)=173.54, P<0.001) and low in symposia (χ2(5)=36.92, P<0.001). Additionally, women chaired fewer symposia (χ2(5)=47.83, P<0.001). Furthermore, men-chaired symposia had significantly fewer women speakers than women-chaired symposia (χ2(1)=56.44, P<0.001). The gender disparities observed here are similar to those in other scientific societies worldwide, urging them to lead actions to pursue gender balance and diversity. Diversity leads not only to fairness but also to higher-quality science.

The Joule heating problem in silver nanowire transparent electrodes.

Silver nanowire transparent electrodes have shown considerable potential to replace conventional transparent conductive materials. However, in this report we show that Joule heating is a unique and serious problem with these electrodes. When conducting current densities encountered in organic solar cells, the average surface temperature of indium tin oxide (ITO) and silver nanowire electrodes, both with sheet resistances of 60 ohms/square, remains below 35 °C. However, in contrast to ITO, the temperature in the nanowire electrode is very non-uniform, with some localized points reaching temperatures above 250 °C. These hotspots accelerate nanowire degradation, leading to electrode failure after 5 days of continuous current flow. We show that graphene, a commonly used passivation layer for these electrodes, slows nanowire degradation and creates a more uniform surface temperature under current flow. However, the graphene does not prevent Joule heating in the nanowires and local points of high temperature ultimately shift the failure mechanism from nanowire degradation to melting of the underlying plastic substrate. In this paper, surface temperature mapping, lifetime testing under current flow, post-mortem analysis, and modelling illuminate the behaviour and failure mechanisms of nanowires under extended current flow and provide guidelines for managing Joule heating.

Long-term expression of melanopsin and channelrhodopsin causes no gross alterations in the dystrophic dog retina.

Several preclinical studies have investigated the potential of algal channelrhodopsin and human melanopsin as optogenetic tools for vision restoration. In the present study, we assessed the potentially deleterious effects of long-term expression of these optogenes on the diseased retina in a large animal model of retinal degeneration, the RPE65-deficient Briard dog model of Leber congenital amaurosis. Intravitreal injection of adeno-associated virus vectors expressing channelrhodopsin and melanopsin had no effect on retinal thickness over a 16-month period post injection. Our data support the safety of the optogenetic approach for the treatment of blindness.

Body mass index, cardiorespiratory fitness and cardiometabolic risk factors in youth from Portugal and Mozambique.

OBJECTIVES: The objectives of this study are to examine differences in cardiometabolic risk indicators, as well as their prevalences, in Portuguese and Mozambican youth, and to investigate the associations between weight status and cardiorespiratory fitness levels with cardiometabolic risk. METHODS: The sample comprises 721 adolescents (323 Mozambican and 398 Portuguese), aged 10-15 years. Anthropometry (height, sitting height, weight and waist circumference), blood pressure, serum-fasting triglycerides, high-density lipoprotein cholesterol and glucose, and cardiorespiratory fitness were measured. Maturity offset was estimated and a cardiometabolic risk score adjusted for sex, age and biological maturity was computed. Adolescents were classified as normal weight and overweight/obese as well as fit or unfit (cardiorespiratory fitness). RESULTS: Portuguese youth have better cardiometabolic and cardiorespiratory fitness profiles. About 32% and 30% of Portuguese boys and girls, respectively, are overweight/obese; in Mozambicans, these prevalences are 7.5% for boys and 21% for girls; in addition, 81.6% of Portuguese boys and 77.7% of Portuguese girls were classified as cardiorespiratory fit, against 54% and 44.4% of Mozambican boys and girls, respectively. No statistically significant differences (P>0.05) were found between Mozambicans and Portuguese for the cluster of three or more cardiometabolic risk indicators. A positive relationship (P<0.001) was found between weight status and cardiometabolic risk in adolescents from both countries; however, a negative association (P<0.001) between cardiorespiratory fitness and cardiometabolic risk was only found among Portuguese youth. CONCLUSIONS: Portuguese and Mozambican youth differ in their cardiometabolic risk profiles, body weight and cardiorespiratory fitness, favoring Portuguese. Overweight/obesity and low cardiorespiratory fitness levels are related to a worse cardiometabolic risk profile, being relevant to design public health intervention strategies to reduce excess weight and increase cardiorespiratory fitness.

Efficient central nervous system AAVrh10-mediated intrathecal gene transfer in adult and neonate rats.

Intracerebral administration of recombinant adeno-associated vector (AAV) has been performed in several clinical trials. However, delivery into the brain requires multiple injections and is not efficient to target the spinal cord, thus limiting its applications. To assess widespread and less invasive strategies, we tested intravenous (IV) or intrathecal (that is, in the cerebrospinal fluid (CSF)) delivery of a rAAVrh10-egfp vector in adult and neonate rats and studied the effect of the age at injection on neurotropism. IV delivery is more efficient in neonates and targets predominantly Purkinje cells of the cerebellum and sensory neurons of the spinal cord and dorsal root ganglia. A single intra-CSF administration of AAVrh10, single strand or oversized self-complementary, is efficient for the targeting of neurons in the cerebral hemispheres, cerebellum, brainstem and spinal cord. Green fluorescent protein (GFP) expression is more widespread in neonates when compared with adults. More than 50% of motor neurons express GFP in the three segments of the spinal cord in neonates and in the cervical and thoracic regions in adults. Neurons are almost exclusively transduced in neonates, whereas neurons, astrocytes and rare oligodendrocytes are targeted in adults. These results expand the possible routes of delivery of AAVrh10, a serotype that has shown efficacy and safety in clinical trials concerning neurodegenerative diseases.

Molecular characteristics of Human Endogenous Retrovirus type-W in schizophrenia and bipolar disorder.

Epidemiological and genome-wide association studies of severe psychiatric disorders such as schizophrenia (SZ) and bipolar disorder (BD), suggest complex interactions between multiple genetic elements and environmental factors. The involvement of genetic elements such as Human Endogenous Retroviruses type 'W' family (HERV-W) has consistently been associated with SZ. HERV-W envelope gene (env) is activated by environmental factors and encodes a protein displaying inflammation and neurotoxicity. The present study addressed the molecular characteristics of HERV-W env in SZ and BD. Hundred and thirty-six patients, 91 with BD, 45 with SZ and 73 healthy controls (HC) were included. HERV-W env transcription was found to be elevated in BD (P<10-4) and in SZ (P=0.012) as compared with HC, but with higher values in BD than in SZ group (P<0.01). The corresponding DNA copy number was paradoxically lower in the genome of patients with BD (P=0.0016) or SZ (P<0.0003) than in HC. Differences in nucleotide sequence of HERV-W env were found between patients with SZ and BD as compared with HC, as well as between SZ and BD. The molecular characteristics of HERV-W env also differ from what was observed in Multiple Sclerosis (MS) and may represent distinct features of the genome of patients with BD and SZ. The seroprevalence for Toxoplasma gondii yielded low but significant association with HERV-W transcriptional level in a subgroup of BD and SZ, suggesting a potential role in particular patients. A global hypothesis of mechanisms inducing such major psychoses is discussed, placing HERV-W at the crossroads between environmental, genetic and immunological factors. Thus, particular infections would act as activators of HERV-W elements in earliest life, resulting in the production of an HERV-W envelope protein, which then stimulates pro-inflammatory and neurotoxic cascades. This hypothesis needs to be further explored as it may yield major changes in our understanding and treatment of severe psychotic disorders.

[Digital ulcers in systemic sclerosis: use of endotheline antagonists]. / Úlceras digitais na esclerodermia: papel dos antagonistas dos receptores da endotelina na terap êutica.

INTRODUCTION: Systemic sclerosis (SSc) is a systemic disease, characterized by fibrosis and vasculopathy, with variable internal organ involvement. Skin is very often involved, namely digital ulcers (DU), seldom treatment resistant, responsible for important functional limitation. The DU can evolve from sclerodactily with superficial ulcers, isquemic lesions, deep necrosis, gangrene, loss of tissue, and consequently, to finger amputation. METHODS: The authors describe the case of a 36 year old female patient, with SSc diagnosed 6 years previously, with skin, lung and gut manifestations. The patient showed uncontrolled Raynaud's phenomenon (RF), despite the adequate treatment using nifedidpine and general local warming measures, with progressively worsening DU and isquemia, especially in cold seasons. Bosentan, 62.5 mg twice daily was started, and a significant improvement in the peripheral isquemic lesions was achieved. The ulcers' healing was fast, the patient totally recovered function and regained quality of life, and no further lesions developed. CONCLUSION: The authors review the RF and DU in SSc, as well as the use of bosentan, an endotheline receptor antagonist, and its indications. Although it is not formally approved, the use of bosentan in SS has shown benefits in reducing the incidence of DU, and despite no influence in the healing process, this drug prevents the development of new lesions.

Oral administration of doxycycline allows tight control of transgene expression: a key step towards gene therapy of retinal diseases.

Gene transfer of neurotrophic or antiangiogenic factors has been shown to improve photoreceptor survival in retinal degenerative disorders (that is retinitis pigmentosa) and to prevent neovascularization in retinal vascular diseases (that is age-related macular degeneration, diabetic retinopathy). Expression of such neurotrophic or antiangiogenic factors after gene transfer requires the use of a regulatory system to control transgene expression to avoid unwanted side effects in cases of overexpression. In a previous study, we demonstrated that rAAV-mediated gene transfer of the tetracycline-regulatable (tetR) system allows transgene regulation in the retina of nonhuman primates after intravenous administration of doxycycline (Dox). The purpose of this study was to evaluate oral administration of Dox to control transgene expression in the retina, since the pharmacokinetics after oral administration of the inducer drug represent a key factor when considering advancing to clinical trials. We report on the outcome of this evaluation and demonstrate that oral administration of Dox at a dose that is clinically used in humans (5 mg kg(-1) per day) is capable to continuously induce transgene expression in all macaques tested for 6 months. Moreover, control of transgene expression persists up to 4 years post-subretinal injection, with maximal induced levels of transgene product remaining stable over time.

Restoration of vision in RPE65-deficient Briard dogs using an AAV serotype 4 vector that specifically targets the retinal pigmented epithelium.

Previous studies have tested gene replacement therapy in RPE65-deficient dogs using recombinant adeno-associated virus 2/2 (rAAV2/2), -2/1 or -2/5 mediated delivery of the RPE65 gene. They all documented restoration of dark- and light-adapted electroretinography responses and improved psychophysical outcomes. Use of a specific RPE65 promoter and a rAAV vector that targets transgene expression specifically to the RPE may, however, provide a safer setting for the long-term therapeutic expression of RPE65. Subretinal injection of rAAV2 pseudotyped with serotype 4 (rAAV2/4) specifically targets the RPE. The purpose of our study was to evaluate a rAAV2/4 vector carrying a human RPE65cDNA driven by a human RPE65 promoter, for the ability to restore vision in RPE65-/- purebred Briard dogs and to assess the safety of gene transfer with respect to retinal morphology and function. rAAV2/4 and rAAV2/2 vectors containing similar human RPE65 promoter and cDNA cassettes were generated and administered subretinally in eight affected dogs, ages 8-30 months (n = 6 with rAAV2/4, n = 2 with rAAV2/2). Although fluorescein angiography and optical coherence tomography examinations displayed retinal abnormalities in treated retinas, electrophysiological analysis demonstrated that restoration of rod and cone photoreceptor function started as soon as 15 days post-injection, reaching maximal function at 3 months post-injection, and remaining stable thereafter in all animals treated at 8-11 months of age. As assessed by the ability of these animals to avoid obstacles in both dim and normal light, functional vision was restored in the treated eye, whereas the untreated contralateral eye served as an internal control. The dog treated at a later age (30 months) did not recover retinal function or vision, suggesting that there might be a therapeutic window for the successful treatment of RPE65-/- dogs by gene replacement therapy.

SET protein (TAF1beta, I2PP2A) is involved in neuronal apoptosis induced by an amyloid precursor protein cytoplasmic subdomain.

When overexpressed, a short cytoplasmic domain of the amyloid precursor protein (APP), normally unmasked in the brain of Alzheimer's disease patients, activates caspase-3 and induces neuronal death. Death induction by this "Jcasp" domain is lost when tyrosine 653 is changed into an aspartate, suggesting specific interactions with unknown partners. To identify these putative partners and start to elucidate the mechanisms involved in Jcasp-induced cell death, we internalized a biotinylated version of the peptide into primary neurons and analyzed intracellular interacting proteins by pull-down and mass spectrometry. We find that SET protein, also called template-activating factor (TAF1beta) or phosphatase 2A inhibitor 2 (I2(PP2A)), specifically binds Jcasp early after internalization and that SET and Jcasp interact directly in vitro. Down-regulation of SET reduces Jcasp-induced cell death, confirming a role of this protein in Jcasp-induced apoptosis. Conversely, SET gain of function increases cell death, which suggests that SET level is crucial for neuronal survival/death. Taken together, these results suggest that SET is part of a neuronal apoptotic pathway related to Alzheimer's disease and provides a new entry in the analysis of this pathology.

In vivo gene transfer to the rat retina using herpes simplex virus type 1 (HSV-1)-based amplicon vectors.

The purpose of our study was to evaluate the transduction profiles of herpes simplex virus type 1 (HSV-1)-based amplicon vectors following subretinal injection in the rat. Two amplicon vectors were tested, pHy-CMVGFP and pHy-RPEGFP, both carrying the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) ubiquitous promoter or the RPE65-specific promoter, respectively. For the two amplicon vectors, the GFP reporter gene was efficiently expressed in retinal pigment epithelial (RPE) cells but not in the adjacent photoreceptors. GFP expression was maximum as early as 2 days post-administration but decreased over time to become almost undetectable at 6 weeks postinjection. Super-transduction with a second amplicon vector, pHSVlac, reactivated expression of GFP in approximately 10% of the cells initially transduced at 2 days postinjection of pHy-CMVGFP or pHy-RPEGFP. Reactivation of transgene expression was transient, no GFP signal was detected 8 days after pHSVlac injection. In conclusion, HSV-1 amplicon vectors allow rapid and efficient, but transient, gene transfer in RPE cells following subretinal injection.

New designs for MRI contrast agents.

New designs for Magnetic Resonance Imaging contrast agents are presented. Essentially, they all are host-guest inclusion complexes between y-cyclodextrins and polyazamacrocycles of gadolinium (III) ion. Substitutions have been made to the host to optimise the host-guest association. Molecular mechanics calculations have been performed, using the UFF force field for metals, to decide on the suitability of the substitutions, and to evaluate the host-guest energies of association. Interesting general conclusions have been obtained, concerning the improvement of Magnetic Resonance Imaging contrast agents; namely, a set of rational methodologies have been deduced to improve the association between the gadolinium (III) chelates and the cyclodextrins, and their efficiency is demonstrated with a large set of substituted complexes, opening new doors to increase the diagnostic capabilities of Magnetic Resonance Imaging.

A multiplex PCR assay for the simultaneous detection and discrimination of the seven Eimeria species that infect domestic fowl.

This study reports the development of a novel multiplex PCR assay based on SCAR (Sequence-Characterised Amplified Region) markers for the simultaneous diagnosis of the 7 Eimeria species that infect domestic fowl. Primer pairs specific for each species were designed in order to generate a ladder of amplification products ranging from 200 to 811 bp. Sensitivity tests for each species were carried out, showing a detection threshold of 1-5 pg, which corresponds approximately to 2-8 sporulated oocysts. Distinct isolates of the 7 Eimeria species from different geographical sources were tested and successfully detected by the assay. All the species were amplified homogeneously, whether or not one of them was present in a high quantity, indicating that there was no cross-interference. The assay was also tested with different sources of Taq DNA polymerase and thermocycler models, confirming the high reproducibility of the reaction. The economy of consumables and labour represented by a single-tube reaction greatly facilitates the molecular diagnosis of a large number of samples, making it appropriate for field epizootiological surveys. We propose the use of this multiplex PCR assay as a rapid and cost-effective diagnostic method for the detection and discrimination of the 7 Eimeria species that infect domestic fowl.

A survey of the inter- and intraspecific RAPD markers of Eimeria spp. of the domestic fowl and the development of reliable diagnostic tools.

Coccidiosis of domestic fowl is a protozoan disease, caused by seven distinct species of the genus Eimeria, which is responsible for important economic losses in poultry production. In order to select RAPD primers for the discrimination of these seven Eimeria species, we carried out an initial screening using samples of E. acervulina, E. tenella and E. maxima. Out of 150 primers tested, 110 generated band profiles specific for each one of these species. A subset of 14 oligonucleotides were also tested for the simultaneous differentiation of the seven species, resulting in 11 discriminative primers. The intraspecific discrimination was assessed for five different species, using samples from different geographic regions including three continents. Numerous primers exhibited highly discriminative band profiles containing strain-specific markers, with a higher variability being observed among strains of E. acervulina than among E. tenella and E. maximastrains. However, no major differences were observed in the band patterns from strains collected in locations near to one another compared to strains originating from distantly located regions. Because RAPD is a technique performed under low stringency conditions, it suffers from poor reproducibility. Aiming at obtaining more reliable markers that might be universally used, we started an effort to convert species-specific RAPD fragments into SCAR markers. An initial conversion of 25 RAPD markers into SCARs, followed by validation of their specificity, resulted in 14 totally new Eimeria species-specific markers that can be used for the molecular diagnosis of the seven species that infect domestic fowl. This work represents a first step in the development of a set of species-specific SCARs that will be useful as tools for molecular diagnosis, genome mapping, and genetic diversity studies.