Insights into the phylogeny of Northern Hemisphere Armillaria: Neighbor-net and Bayesian analyses of translation elongation factor 1-α gene sequences.

Armillaria possesses several intriguing characteristics that have inspired wide interest in understanding phylogenetic relationships within and among species of this genus. Nuclear ribosomal DNA sequence-based analyses of Armillaria provide only limited information for phylogenetic studies among widely divergent taxa. More recent studies have shown that translation elongation factor 1-α (tef1) sequences are highly informative for phylogenetic analysis of Armillaria species within diverse global regions. This study used Neighbor-net and coalescence-based Bayesian analyses to examine phylogenetic relationships of newly determined and existing tef1 sequences derived from diverse Armillaria species from across the Northern Hemisphere, with Southern Hemisphere Armillaria species included for reference. Based on the Bayesian analysis of tef1 sequences, Armillaria species from the Northern Hemisphere are generally contained within the following four superclades, which are named according to the specific epithet of the most frequently cited species within the superclade: (i) Socialis/Tabescens (exannulate) superclade including Eurasian A. ectypa, North American A. socialis (A. tabescens), and Eurasian A. socialis (A. tabescens) clades; (ii) Mellea superclade including undescribed annulate North American Armillaria sp. (Mexico) and four separate clades of A. mellea (Europe and Iran, eastern Asia, and two groups from North America); (iii) Gallica superclade including Armillaria Nag E (Japan), multiple clades of A. gallica (Asia and Europe), A. calvescens (eastern North America), A. cepistipes (North America), A. altimontana (western USA), A. nabsnona (North America and Japan), and at least two A. gallica clades (North America); and (iv) Solidipes/Ostoyae superclade including two A. solidipes/ostoyae clades (North America), A. gemina (eastern USA), A. solidipes/ostoyae (Eurasia), A. cepistipes (Europe and Japan), A. sinapina (North America and Japan), and A. borealis (Eurasia) clade 2. Of note is that A. borealis (Eurasia) clade 1 appears basal to the Solidipes/Ostoyae and Gallica superclades. The Neighbor-net analysis showed similar phylogenetic relationships. This study further demonstrates the utility of tef1 for global phylogenetic studies of Armillaria species and provides critical insights into multiple taxonomic issues that warrant further study.

A Diverse Soil Microbiome Degrades More Crude Oil than Specialized Bacterial Assemblages Obtained in Culture.

UNLABELLED: Soil microbiome modification may alter system function, which may enhance processes like bioremediation. In this study, we filled microcosms with gamma-irradiated soil that was reinoculated with the initial soil or cultivated bacterial subsets obtained on regular media (REG-M) or media containing crude oil (CO-M). We allowed 8 weeks for microbiome stabilization, added crude oil and monoammonium phosphate, incubated the microcosms for another 6 weeks, and then measured the biodegradation of crude oil components, bacterial taxonomy, and functional gene composition. We hypothesized that the biodegradation of targeted crude oil components would be enhanced by limiting the microbial taxa competing for resources and by specifically selecting bacteria involved in crude oil biodegradation (i.e., CO-M). Postincubation, large differences in taxonomy and functional gene composition between the three microbiome types remained, indicating that purposeful soil microbiome structuring is feasible. Although phylum-level bacterial taxonomy was constrained, operational taxonomic unit composition varied between microbiome types. Contrary to our hypothesis, the biodegradation of C10 to C50 hydrocarbons was highest when the original microbiome was reinoculated, despite a higher relative abundance of alkane hydroxylase genes in the CO-M microbiomes and of carbon-processing genes in the REG-M microbiomes. Despite increases in the relative abundances of genes potentially linked to hydrocarbon processing in cultivated subsets of the microbiome, reinoculation of the initial microbiome led to maximum biodegradation. IMPORTANCE: In this study, we show that it is possible to sustainably modify microbial assemblages in soil. This has implications for biotechnology, as modification of gut microbial assemblages has led to improved treatments for diseases like Clostridium difficile infection. Although the soil environment determined which major phylogenetic groups of bacteria would dominate the assemblage, we saw differences at lower levels of taxonomy and in functional gene composition (e.g., genes related to hydrocarbon degradation). Further studies are needed to determine the success of such an approach in nonsterile environments. Although the biodegradation of certain crude oil fractions was still the highest when we inoculated with the diverse initial microbiome, the possibility of discovering and establishing microbiomes that are more efficient in crude oil degradation is not precluded.

The large (134.9 kb) mitochondrial genome of the glomeromycete Funneliformis mosseae.

Funneliformis mosseae is among the most ecologically and economically important glomeromycete species and occurs both in natural and disturbed areas in a wide range of habitats and climates. In this study, we report the sequencing of the complete mitochondrial (mt) genome of F. mosseae isolate FL299 using 454 pyrosequencing and Illumina HiSeq technologies. This mt genome is a full-length circular chromosome of 134,925 bp, placing it among the largest mitochondrial DNAs (mtDNAs) in the fungal kingdom. A comparative analysis with publically available arbuscular mycorrhizal fungal mtDNAs revealed that the mtDNA of F. mosseae FL299 contained a very large number of insertions contributing to its expansion. The gene synteny was completely reshuffled compared to previously published glomeromycotan mtDNAs and several genes were oriented in an anti-sense direction. Furthermore, the presence of different types of introns and insertions in rnl (14 introns) made this gene very distinctive in Glomeromycota. The presence of alternative genetic codes in both initiation (GUG) and termination (UGA) codons was another new feature in this mtDNA compared to previously published glomeromycotan mt genomes. The phylogenetic analysis inferred from the analysis of 14 protein mt genes confirmed the position of the Glomeromycota clade as a sister group of Mortierellomycotina. This mt genome is the largest observed so far in Glomeromycota and the first mt genome within the Funneliformis clade, providing new opportunities to better understand their evolution and to develop molecular markers.

Molecular diagnostic toolkit for Rhizophagus irregularis isolate DAOM-197198 using quantitative PCR assay targeting the mitochondrial genome.

Rhizophagus irregularis (previously named Glomus irregulare) is one of the most widespread and common arbuscular mycorrhizal fungal (AMF) species. It has been recovered worldwide in agricultural and natural soils, and the isolate DAOM-197198 has been utilized as a commercial inoculant for two decades. Despite the ecological and economical importance of this taxon, specific markers for quantification of propagules by quantitative real-time PCR (qPCR) are extremely limited and none have been rigorously validated for quality control of manufactured products such as biofertilizers. From the sequencing of 14 complete AMF mitochondrial (mt) genomes, a qPCR assay using a hydrolysis probe designed in the single copy cox3-rnl intergenic region was tested and validated to specifically and accurately quantify the spores of R. irregularis isolate DAOM-197198. Specificity tests were performed using standard PCR and qPCR, and results clearly showed that the primers specifically amplified the isolate DAOM-197198, yielding a PCR product of 106 bp. According to the qPCR analyses on spores produced in vitro, the average copy number of mt genomes per spore was 3172 ± 304 SE (n = 6). Quantification assays were successfully undertaken on known and unknown samples in liquid suspensions and commercial dry formulations to show the accuracy, precision, robustness, and reproducibility of the qPCR assay. This study provides a powerful molecular toolkit specifically designed to quantify spores of the model AMF isolate DAOM-197198. The approach of molecular toolkit used in our study could be applied to other AMF taxa and will be useful to research institutions and governmental and industrial laboratories running routine quality control of AMF-based products.

Culture-Dependent and -Independent Methods Capture Different Microbial Community Fractions in Hydrocarbon-Contaminated Soils.

Bioremediation is a cost-effective and sustainable approach for treating polluted soils, but our ability to improve on current bioremediation strategies depends on our ability to isolate microorganisms from these soils. Although culturing is widely used in bioremediation research and applications, it is unknown whether the composition of cultured isolates closely mirrors the indigenous microbial community from contaminated soils. To assess this, we paired culture-independent (454-pyrosequencing of total soil DNA) with culture-dependent (isolation using seven different growth media) techniques to analyse the bacterial and fungal communities from hydrocarbon-contaminated soils. Although bacterial and fungal rarefaction curves were saturated for both methods, only 2.4% and 8.2% of the bacterial and fungal OTUs, respectively, were shared between datasets. Isolated taxa increased the total recovered species richness by only 2% for bacteria and 5% for fungi. Interestingly, none of the bacteria that we isolated were representative of the major bacterial OTUs recovered by 454-pyrosequencing. Isolation of fungi was moderately more effective at capturing the dominant OTUs observed by culture-independent analysis, as 3 of 31 cultured fungal strains ranked among the 20 most abundant fungal OTUs in the 454-pyrosequencing dataset. This study is one of the most comprehensive comparisons of microbial communities from hydrocarbon-contaminated soils using both isolation and high-throughput sequencing methods.

Arbuscular mycorrhizal fungal diversity associated with Eleocharis obtusa and Panicum capillare growing in an extreme petroleum hydrocarbon-polluted sedimentation basin.

Arbuscular mycorrhizal fungi (AMF) have been extensively studied in natural and agricultural ecosystems, but little is known about their diversity and community structure in highly petroleum-polluted soils. In this study, we described an unexpected diversity of AMF in a sedimentation basin of a former petrochemical plant, in which petroleum hydrocarbon (PH) wastes were dumped for many decades. We used high-throughput PCR, cloning and sequencing of 18S rDNA to assess the molecular diversity of AMF associated with Eleocharis obtusa and Panicum capillare spontaneously inhabiting extremely PH-contaminated sediments. The analyses of rhizosphere and root samples over two years showed a remarkable AMF richness comparable with that found in temperate natural ecosystems. Twenty-one taxa, encompassing the major families within Glomeromycota, were detected. The most abundant OTUs belong to genera Claroideoglomus, Diversispora, Rhizophagus and Paraglomus. Both plants had very similar overall community structures and OTU abundances; however, AMF community structure differed when comparing the overall OTU distribution across the two years of sampling. This could be likely explained by variations in precipitations between 2011 and 2012. Our study provides the first view of AMF molecular diversity in soils extremely polluted by PH, and demonstrated the ability of AMF to colonize and establish in harsh environments.

The mitochondrial genome of the glomeromycete Rhizophagus sp. DAOM 213198 reveals an unusual organization consisting of two circular chromosomes.

Mitochondrial (mt) genomes are intensively studied in Ascomycota and Basidiomycota, but they are poorly documented in basal fungal lineages. In this study, we sequenced the complete mtDNA of Rhizophagus sp. DAOM 213198, a close relative to Rhizophagus irregularis, a widespread, ecologically and economical relevant species belonging to Glomeromycota. Unlike all other known taxonomically close relatives harboring a full-length circular chromosome, mtDNA of Rhizophagus sp. reveals an unusual organization with two circular chromosomes of 61,964 and 29,078 bp. The large chromosome contained nine protein-coding genes (atp9, nad5, cob, nad4, nad1, nad4L, cox1, cox2, and atp8), small subunit rRNA gene (rns), and harbored 20 tRNA-coding genes and 10 orfs, while the small chromosome contained five protein-coding genes (atp6, nad2, nad3, nad6, and cox3), large subunit rRNA gene (rnl) in addition to 5 tRNA-coding genes, and 8 plasmid-related DNA polymerases (dpo). Although structural variation of plant mt genomes is well documented, this study is the first report of the presence of two circular mt genomes in arbuscular mycorrhizal fungi. Interestingly, the presence of dpo at the breakage point in intergenes cox1-cox2 and rnl-atp6 for large and small mtDNAs, respectively, could be responsible for the conversion of Rhizophagus sp. mtDNA into two chromosomes. Using quantitative real-time polymerase chain reaction, we found that both mtDNAs have an equal abundance. This study reports a novel mtDNA organization in Glomeromycota and highlights the importance of studying early divergent fungal lineages to describe novel evolutionary pathways in the fungal kingdom.

Contrasting the community structure of arbuscular mycorrhizal fungi from hydrocarbon-contaminated and uncontaminated soils following willow (Salix spp. L.) planting.

Phytoremediation is a potentially inexpensive alternative to chemical treatment of hydrocarbon-contaminated soils, but its success depends heavily on identifying factors that govern the success of root-associated microorganisms involved in hydrocarbon degradation and plant growth stimulation. Arbuscular mycorrhizal fungi (AMF) form symbioses with many terrestrial plants, and are known to stimulate plant growth, although both species identity and the environment influence this relationship. Although AMF are suspected to play a role in plant adaptation to hydrocarbon contamination, their distribution in hydrocarbon-contaminated soils is not well known. In this study, we examined how AMF communities were structured within the rhizosphere of 11 introduced willow cultivars as well as unplanted controls across uncontaminated and hydrocarbon-contaminated soils at the site of a former petrochemical plant. We obtained 69 282 AMF-specific 18S rDNA sequences using 454-pyrosequencing, representing 27 OTUs. Contaminant concentration was the major influence on AMF community structure, with different AMF families dominating at each contaminant level. The most abundant operational taxonomic unit in each sample represented a large proportion of the total community, and this proportion was positively associated with increasing contamination, and seemingly, by planting as well. The most contaminated soils were dominated by three phylotypes closely related to Rhizophagus irregularis, while these OTUs represented only a small proportion of sequences in uncontaminated and moderately contaminated soils. These results suggest that in situ inoculation of AMF strains could be an important component of phytoremediation treatments, but that strains should be selected from the narrow group that is both adapted to contaminant toxicity and able to compete with indigenous AMF species.

Concordance of seven gene genealogies compared to phenotypic data reveals multiple cryptic species in Australian dermocyboid Cortinarius (Agaricales).

This study aims to delimit species of Australian dermocyboid fungi (Cortinarius, Agaricales) using genealogical concordance on well-characterised phenotypic species and to assess the utility of seven loci for DNA barcoding Australian Cortinarius taxa. Eighty-six collections of dermocyboid Cortinarius were sampled from across southern Australia. Phenotypic species were first recognised by performing clustering analyses on a comprehensive phenotypic dataset including morphological, colour and pigment data. Then phylogenetic species were delimited from the concordance of seven locus genealogies (ITS, nLSU, gpd, mcm7, rpb1, rpb2 and tef1). Seventeen phenotypic species were recognised while the concordance of gene genealogies recovered 35 phylogenetic species. All loci except for LSU recovered most phylogenetic species, although only rpb1 correctly identified all phylogenetic species. The ITS region is confirmed as an effective barcode for Cortinarius and a standard pairwise distance threshold of 2.0% is proposed to DNA barcode Australian Cortinarius taxa. Australian dermocyboid fungi belong in separate clades to the boreal clade Dermocybe, mostly in the clade Splendidi. This study provides a solid foundation for future ecological, taxonomic and systematic research on one of the most diverse genera of mushrooms worldwide.

Impact of endochitinase-transformed white spruce on soil fungal communities under greenhouse conditions.

Chitinase genes isolated from plants, bacteria or fungi have been widely used in genetic engineering to enhance the resistance of crops and trees to fungal pathogens. However, there are concerns about the possible effect of chitinase-transformed plants on nontarget fungi. This study aimed at evaluating the impact of endochitinase-transformed white spruce on soil fungal communities. Endochitinase-expressing white spruce and untransformed controls were transplanted in soils from two natural forests and grown for 8 months in a greenhouse. Soil fungal biomass and diversity, estimated through species richness and Shannon and Rao diversity indices, were not different between transgenic and control tree rhizospheres. The fungal phylogenetic community structure was the same in soil samples from control and transgenic white spruces after 8 months. Soil type and presence of seedlings had a much more significant impact on fungal community structure than the insertion and expression of the ech42 transgene within the white spruce genome. The results suggest that the insertion and constitutive expression of the ech42 gene in white spruce did not significantly affect soil fungal biomass, diversity and community structure.

Morchella tomentosa: a unique belowground structure and a new clade of morels.

Mechanisms involved in post-fire morel fructification remain unclear. A new undescribed belowground vegetative structure of Morchella tomentosa in a burned boreal forest was investigated north of Fairbanks, Alaska. The name "radiscisclerotium" is proposed to define this peculiar and elaborate below-ground vegetative structure of M. tomentosa. Bayesian and maximum parsimony analyses based on ITS rRNA regions and nLSU gene strongly supported a new clade composed of M. tomentosa within the genus Morchella.

Impact of endochitinase-transformed white spruce on soil fungal biomass and ectendomycorrhizal symbiosis.

The impact of transgenic white spruce [Picea glauca (Moench) Voss] containing the endochitinase gene (ech42) on soil fungal biomass and on the ectendomycorrhizal fungi Wilcoxina spp. was tested using a greenhouse trial. The measured level of endochitinase in roots of transgenic white spruce was up to 10 times higher than that in roots of nontransformed white spruce. The level of endochitinase in root exudates of three of four ech42-transformed lines was significantly greater than that in controls. Analysis soil ergosterol showed that the amount of fungal biomass in soil samples from control white spruce was slightly larger than that in soil samples from ech42-transformed white spruce. Nevertheless, the difference was not statistically significant. The rates of mycorrhizal colonization of transformed lines and controls were similar. Sequencing the internal transcribed spacer rRNA region revealed that the root tips were colonized by the ectendomycorrhizal fungi Wilcoxina spp. and the dark septate endophyte Phialocephala fortinii. Colonization of root tips by Wilcoxina spp. was monitored by real-time PCR to quantify the fungus present during the development of ectendomycorrhizal symbiosis in ech42-transformed and control lines. The numbers of Wilcoxina molecules in the transformed lines and the controls were not significantly different (P > 0.05, as determined by analysis of covariance), indicating that in spite of higher levels of endochitinase expression, mycorrhization was not inhibited. Our results indicate that the higher levels of chitinolytic activity in root exudates and root tissues from ech42-transformed lines did not alter the soil fungal biomass or the development of ectendomycorrhizal symbiosis involving Wilcoxina spp.

Comparing ballistic wounds with experiments on body simulator.

This paper demonstrates how ballistic experiments on body simulator can bring a key information in the forensic science field. In the investigated case, a hunter was shot by accident in the back. Two hunters were suspected of having inadvertently shot towards the victim. The deadly bullet left the body and cannot be found on the scene neither in the body. The only way to discriminate the two options was to perform ballistic tests in body simulators. Even though the knowledge about body simulators is not enough advanced yet to expect accurate quantitative results, it was supposed to fully discriminate the two investigated cases as its respective impact energy are highly different (respectively 1200J and 2400J). For each investigated possibility, bullet's expansion state and body wounds were simulated. Bullet impact characteristics were determined by measuring the muzzle velocity, compute the impact velocity in the considered range (the position of each hunter is accurately known). Reloading cartridges allowed to reproduce accuretaly the corresponding velocity. The body was simulated by 3 different means in order to explore the accuracy of the simulation process. We demonstrated that the reported case is situated in a velocity/energy range in which body simulators do not need to be particularly accurate to reproduce the bullet expansion/non-expansion state. It furthermore demonstrated that only one case is compatible with the ballistic wounds of the victim. In the other case, the bullet's expansion would lead to a completely different wound shape.

Impact of an 8-year-old transgenic poplar plantation on the ectomycorrhizal fungal community.

The long-term impact of field-deployed genetically modified trees on soil mutualistic organisms is not well known. This study aimed at evaluating the impact of poplars transformed with a binary vector containing the selectable nptII marker and beta-glucuronidase reporter genes on ectomycorrhizal (EM) fungi 8 years after field deployment. We generated 2,229 fungal internal transcribed spacer (ITS) PCR products from 1,150 EM root tips and 1,079 fungal soil clones obtained from the organic and mineral soil horizons within the rhizosphere of three control and three transformed poplars. Fifty EM fungal operational taxonomic units were identified from the 1,706 EM fungal ITS amplicons retrieved. Rarefaction curves from both the root tips and soil clones were close to saturation, indicating that most of the EM species present were recovered. Based on qualitative and/or quantitative alpha- and beta-diversity measurements, statistical analyses did not reveal significant differences between EM fungal communities associated with transformed poplars and the untransformed controls. However, EM communities recovered from the root tips and soil cloning analyses differed significantly from each other. We found no evidence of difference in the EM fungal community structure linked to the long-term presence of the transgenic poplars studied, and we showed that coupling root tip analysis with a soil DNA cloning strategy is a complementary approach to better document EM fungal diversity.

Estimating the shooting distance of a 9-mm Parabellum bullet via ballistic experiment.

We demonstrate here how the shooting distance of a 9-mm Parabellum FMJ bullet (115gr) has been estimated via shooting experiments. Such a bullet was found by investigators near a concrete wall, fairly distorted at its tip. The bullet carries no evidence of multiple impact and no evidence of ballistic impact on the wall has been reported. We estimated the impact velocity by comparing the questioned bullet with a set of comparison bullets hitting a wall (rigid target) with different velocities. The shooting distance was recovered from the impact velocity by studying the typical behavior of a manufactured 9 mm bullet weighting 115g (7.45g), shot in pistol or a sub-machine gun. The results demonstrated that the questioned bullet was a lost bullet. The shooting distance also helped the investigators, narrowing the range of the estimated positions of the shooter.

[Amniotic membrane graft in ocular surface disease. Prospective study with 31 cases]. / La greffe de membrane amniotique dans les pathologies oculaires de surface. Etude prospective à partir de 31 cas.

INTRODUCTION: Amniotic membrane's unique combination of properties including the facilitation of migration of epithelial cells, the reinforcement of basal cellular adhesion and the encouragement of epithelial differentiation [6] together with its ability to modulate stromal scarring and its anti-inflammatory and anti-bacterial activity has led to its use in the treatment of ocular surface pathology as well as an adjunct to stem cell grafts of the corneal limbus [6-4]. We report a prospective study of 30 patients so treated. MATERIAL AND METHODS: We studied 31 eyes of 30 patients subjected to amniotic membrane grafts between September 1999 and May 2000. There were 25 men and 5 women with an average age of 60.1 (range 25-86) years who were followed for a mean of 7.7 (range 4-11) months. 5 groups (A to D) were observed: A: 6 eyes. Small chronic ulcers without limbal involvement. B: 4 eyes. Ulcers of at least 75% corneal area or occupying 75% of the limbus. C: 9 eyes. Corneal burns. D: 8 eyes. Painful bullous corneal dystrophies unresponsive to other treatment. E: 4 eyes. Symblepharons. Amniotic membrane was placed on the corneal lesion, epithelial surface externally [6, 15], trimmed and sutured with interrupted 10/0 nylon, removed at one month. In two patients (11, 12) inflamed conjunctiva was recessed and amnion sutured to the recessed margin. For the bullous dystrophies we removed all the corneal epithelium and either sutured the amnion to peri-limbal conjunctiva (4 eyes) or to the limbus (4 eyes). For the symblepharons the conjunctiva was dissected to reform the fornix which was lined with amniotic membrane, sutured with 8/0 vicryl. Patients were reviewed regularity. RESULTS: Group A: All healed within 15 days, in most with dissolution of the amnion over 2-3 months although some persisted, covered with corneal epithelium. An eye with a Descemetocoele and one with a microperforation both healed. Vision improved more than two lines in 4 of 6 eyes. Group B: 2 of 4 eyes healed, one despite detachment of the membrane after 15 days. One eye was salvaged by tarsorrhaphy over a fresh keratoplasty after perforation of a neuroparalytic ulcer on failure of three successive amnion grafts. The final cornea vascularised despite an amnion graft for a meta-herpetic ulcer. Group C: 2 of 9 eyes had limbal damage in one quadrant but 7 had vessels in at least three-quarters of the circumference. One (15) also had a limbal autograft. 3 of 9 eyes healed satisfactorily with more than 2/10 improvement in acuity in each case. 2 showed further neovascularisation despite surface healing. One old chemical burn healed satisfactorily but vascularisation remained 5 eyes failed to heal with lysis of the graft, the patient who had a limbal autograft developed a vascular pannus, and in 4 eyes neovascularisation progressed to cover the entire cornea. Group D: 3 eyes settled with loss of symptoms but in 5 the graft detached within 15 days. All eyes where the membrane had been sutured to the conjunctiva beyond the limbus failed whilst 3 of 4 in which it had been sutured anterior to the limbus succeeded, leaving a persistent whitish membrane under the epithelium. Group E: We were able to reconstruct the cul de sac in 3 out of 4 eyes. In one patient with recurrent pterygium good ocular movement was restored, previously limited by scarring. One with associated ocular surface damage from a thermal burn failed by scarring of the cul de sac a month after surgery. DISCUSSION: Our best results were in persistent trophic ulcers of the cornea (Groups A and B) with a success rate of 80%, comparable to those of others [49, 37, 38]. The ready availability of amniotic membrane in our facility makes amniotic membrane transplantation the main secondary treatment for such lesions, especially because of the visual improvement we obtained. Because we did not observe any improvement in corneal thickness after this treatment we advise its early use before significant stromal lysis. The technique was not sufficient to control the effect of corneal anaesthesia in two eyes [40] or in chemical burns suggesting that amniotic membrane alone is insufficient to promote corneal healing in the absence of limbal stem cells. Nevertheless, three eyes did benefit. It has been suggested [13] that the anti-apoptotic function of amnion may prevent stem cell loss in such eyes [42], thus it appears logical to offer an amniotic membrane graft first, before stem cell transplantation, which may entrain complications in the donor eye if autografted [43] or because of the rejection risk of an allograft. It may be that an amniotic membrane graft simply becomes a holding procedure allowing time to settle the eye so as to allow secondary procedures to address the underlying cause of further damage. Our treatment of bullous dystrophy only succeeded on confining the graft to within the limbus, 3 out of 4 eyes becoming comfortable. By contrast we found amniotic membrane helpful in reconstructing symblepharons in the absence of local inflammation. CONCLUSION: Amniotic membrane grafting is a simple and straightforward surgical technique which should form part of the therapeutic arsenal for the treatment of ocular surface disease. Indications for the technique need further clarification for it is evident that it cannot correct all secondary pathology associated with limbal destruction. It is certainly preferable to conjunctival advancement and has proved useful in the reconstruction of the cul-de-sac.

Sequence and functional comparison in the Beckwith-Wiedemann region: implications for a novel imprinting centre and extended imprinting.

The clustered organization of most imprinted genes in mammals suggests coordinated genetic and epigenetic control mechanisms. Comparisons between human and mouse will help in elucidating these mechanisms by identifying structural and functional similarities. Previously we reported on such a comparison in the central part of the mouse imprinting cluster on distal chromosome 7 with the homologous Beckwith-Wiedemann syndrome (BWS) gene cluster on human chromosome 11p15.5. Here we focus on the adjacent sequences of 0.5 Mb including the KCNQ1/Kcnq1 and CDKN1C/Cdkn1c genes, which are implicated in BWS, and on one of the proposed boundary regions of the imprinting cluster. As in the previously analysed central region, this part of the cluster exhibits a highly conserved arrangement and structure of genes. The most striking similarity is found in the 3' part of the KCNQ1/Kcnq1 genes in large stretches of mostly non-coding sequences. The conserved region includes the recently identified KCNQ1OT1/Kcnq1ot1 antisense transcripts, flanked by a strikingly conserved cluster of LINE/Line elements and a CpG island which we show to carry a maternal germline methylation imprint. This region is likely to be the proposed second imprinting centre (IC2) in the BWS cluster. We also identified several novel genes inside and outside the previously proposed boundaries of the imprinting cluster. One of the genes outside the cluster, Obph1, is imprinted in mouse placenta indicating that at least in extra-embryonic tissues the imprinting cluster extends into a larger domain.

[Technique of harvesting and preparation of amniotic membranes]. / Technique de prélèvement et de préparation des membranes amniotiques.

Amniotic membrane transplantation is a promising surgical technique for clinical management of ocular surface diseases. According to the first published results, the ease with which this highly available tissue can be prepared and preserved makes this technique very attractive. The aim of our study is to report procurement procedures and the preparation technique for amniotic membrane in accordance with the French tissue transplantation legislation.

Sequence conservation and variability of imprinting in the Beckwith-Wiedemann syndrome gene cluster in human and mouse.

In human and mouse most imprinted genes are arranged in chromosomal clusters. This linked organization suggests coordinated mechanisms controlling imprinted expression. We have sequenced 250 kb in the centre of the mouse imprinting cluster on distal chromosome 7 and compared it with the orthologous Beckwith-Wiedemann gene cluster on human chromosome 11p15.5. This first comparative imprinting cluster analysis revealed a high structural and functional conservation of the six orthologous genes identified. However, several striking differences were also discovered. First, compared with the mouse the human sequence is approximately 40% longer, mostly due to insertions of two large repetitive clusters. One of these clusters encompasses an additional gene coding for a homologue of the ribosomal protein L26. Second, pronounced blocks of unique direct repeats characteristic of imprinted genes were only found in the human sequence. Third, two of the orthologous gene pairs Tssc4/TSSC4 and Ltrpc5/LTRPC5 showed apparent differences in imprinting between human and mouse, whereas others like Tssc6/TSSC6 were not imprinted in either organism. Together these results suggest a significant functional and structural variability in the centre of the imprinting cluster. Some genes escape imprinting in both organisms whereas others exhibit tissue- and species-specific imprinting. Hence the control of imprinting in the cluster appears to be a highly dynamic process under fast evolutionary adaptation. Intriguingly, whereas imprinted genes within the cluster contain CpG islands the non-imprinted Ltrpc5 and Tssc6/TSSC6 do not. This and additional comparisons with other imprinted and non-imprinted regions suggest that CpG islands are key features of imprinted domains.

Syntenic organization of the mouse distal chromosome 7 imprinting cluster and the Beckwith-Wiedemann syndrome region in chromosome 11p15.5.

In human and mouse, most imprinted genes are arranged in chromosomal clusters. Their linked organization suggests co-ordinated mechanisms controlling imprinting and gene expression. The identification of local and regional elements responsible for the epigenetic control of imprinted gene expression will be important in understanding the molecular basis of diseases associated with imprinting such as Beckwith-Wiedemann syndrome. We have established a complete contig of clones along the murine imprinting cluster on distal chromosome 7 syntenic with the human imprinting region at 11p15.5 associated with Beckwith-Wiedemann syndrome. The cluster comprises approximately 1 Mb of DNA, contains at least eight imprinted genes and is demarcated by the two maternally expressed genes Tssc3 (Ipl) and H19 which are directly flanked by the non-imprinted genes Nap1l4 (Nap2) and Rpl23l (L23mrp), respectively. We also localized Kcnq1 (Kvlqt1) and Cd81 (Tapa-1) between Cdkn1c (p57(Kip2)) and Mash2. The mouse Kcnq1 gene is maternally expressed in most fetal but biallelically transcribed in most neonatal tissues, suggesting relaxation of imprinting during development. Our findings indicate conserved control mechanisms between mouse and human, but also reveal some structural and functional differences. Our study opens the way for a systematic analysis of the cluster by genetic manipulation in the mouse which will lead to animal models of Beckwith-Wiedemann syndrome and childhood tumours.