Structure of Adeno-Associated Virus Vector DNA following Transduction of the Skeletal Muscle

The skeletal muscle provides a very permissive physiological environment for adeno-associated virus (AAV) type 2-mediated gene transfer. We have studied the early steps leading to the establishment of permanent transgene expression, after injection of recombinant AAV (rAAV) particles in the quadriceps muscle of mice. The animals received an rAAV encoding a secreted protein, murine erythropoietin (mEpo), under the control of the human cytomegalovirus major immediate-early promoter and were sacrificed between 1 and 60 days after injection. The measurement of plasma Epo levels and of hematocrits indicated a progressive increase of transgene expression over the first 2 weeks, followed by a stabilization at maximal plateau values. The rAAV sequences were analyzed by Southern blotting following neutral or alkaline gel electrophoresis of total DNA from injected muscles. While a high number of rAAV sequences were detected during the first 5 days following the injection, only a few percent of these sequences was retained in the animals analyzed after 2 weeks, in which transgene expression was maximal. Double-stranded DNA molecules resulting from de novo second-strand synthesis were detected as early as day 1, indicating that this crucial step of AAV-mediated gene transfer is readily accomplished in the muscle. The templates driving stable gene expression at later time points are low in copy number and structured as high-molecular-weight concatemers or interlocked circles. The presence of the circular form of the rAAV genomes at early time points suggests that the molecular transformations involved in the formation of stable concatemers may involve a rolling-circle type of DNA replication.     

Should the management of radiation therapy for breast cancer be standardized? Results of a survey on current French practices in breast radiotherapy

BackgroundBreast cancer is the most frequent cancer in women in France. Its management has evolved considerably in recent years with a focus on reducing iatrogenic toxicity. The radiotherapy indications are validated in multidisciplinary consultation meetings; however, questions remain outstanding, particularly regarding hypofractionated radiotherapy, partial breast irradiation, and irradiation of the internal mammary chain and axillary lymph node area.Materials and methodsAn online survey was sent to 47 heads of radiotherapy departments in France. The survey consisted of 22 questions concerning indications for irradiation of the supraclavicular, internal mammary and axillary lymph node areas; irradiation techniques and modalities; prescribed doses; and fractionation.ResultsTwenty-four out of 47 centers responded (response rate of 51%). This survey demonstrated a wide variation in the prescribed dose regimen, monoisocentric radiotherapy, and indications of irradiation of the lymph node areas.ConclusionThis survey provides insight into the current radiotherapy practice for breast cancer in France. It shows the need to standardize practices.     

6-Nitro-2,3-dihydroimidazo[2,1-b][1,3]thiazoles: Facile synthesis and comparative appraisal against tuberculosis and neglected tropical diseases

As part of a quest for backups to the antitubercular drug pretomanid (PA-824), we investigated the unexplored 6-nitro-2,3-dihydroimidazo[2,1-b][1,3]-thiazoles and related -oxazoles. The nitroimidazothiazoles were prepared in high yield from 2-bromo-4-nitroimidazole via heating with substituted thiiranes and diisopropylethylamine. Equivalent examples of these two structural classes provided broadly comparable MICs, with 2-methyl substitution and extended aryloxymethyl side chains preferred; albeit, S-oxidised thiazoles were ineffective for tuberculosis. Favourable microsomal stability data for a biaryl thiazole (45) led to its assessment in an acute Mycobacterium tuberculosis mouse model, alongside the corresponding oxazole (48), but the latter proved to be more efficacious. In vitro screening against kinetoplastid diseases revealed that nitroimidazothiazoles were inactive versus leishmaniasis but showed interesting activity, superior to that of the nitroimidazooxazoles, against Chagas disease. Overall, “thio-delamanid” (49) is regarded as the best lead.     

Genotyping-by-sequencing highlights original diversity patterns within a European collection of 1191 maize flint lines,as compared to the maize USDA genebank

Key message

Genotyping by sequencing is suitable for analysis of global diversity in maize. We showed the distinctiveness of flint maize inbred lines of interest to enrich the diversity of breeding programs.

Abstract

Genotyping-by-sequencing (GBS) is a highly cost-effective procedure that permits the analysis of large collections of inbred lines. We used it to characterize diversity in 1191 maize flint inbred lines from the INRA collection, the European Cornfed association panel, and lines recently derived from landraces. We analyzed the properties of GBS data obtained with different imputation methods, through comparison with a 50 K SNP array. We identified seven ancestral groups within the Flint collection (dent, Northern flint, Italy, Pyrenees–Galicia, Argentina, Lacaune, Popcorn) in agreement with breeding knowledge. Analysis highlighted many crosses between different origins and the improvement of flint germplasm with dent germplasm. We performed association studies on different agronomic traits, revealing SNPs associated with cob color, kernel color, and male flowering time variation. We compared the diversity of both our collection and the USDA collection which has been previously analyzed by GBS. The population structure of the 4001 inbred lines confirmed the influence of the historical inbred lines (B73, A632, Oh43, Mo17, W182E, PH207, and Wf9) within the dent group. It showed distinctly different tropical and popcorn groups, a sweet-Northern flint group and a flint group sub-structured in Italian and European flint (Pyrenees–Galicia and Lacaune) groups. Interestingly, we identified several selective sweeps between dent, flint, and tropical inbred lines that co-localized with SNPs associated with flowering time variation. The joint analysis of collections by GBS offers opportunities for a global diversity analysis of maize inbred lines.

     

Spatial genetic structure of <Emphasis Type="Italic">Lissotriton helveticus L</Emphasis>. following the restoration of a forest ponds network

Preserving amphibian genetic diversity through ecological restoration and conservation actions is a major challenge since their populations are declining worldwide. We studied the genetic diversity and spatial genetic structure of the palmate newt (Lissotriton helveticus) 2 years after the restoration of a pond network in northwestern France with the aim of reconstructing fine-scale genetic structure and patterns of colonization. We sampled newts from 29 forest ponds including both restored and non-degraded reference ponds, and genotyped 391 individuals at 12 microsatellite loci. We used two Bayesian clustering methods to spatially delineate genetic clusters, and we also detected potential recent migrants within the network. All ponds showed low levels of observed heterozygosity (Ho?=?0.534) and a mean F _IS of 0.251, possibly indicating a Wahlund or bottleneck effect. Pairwise F _ST suggested limited evidence of genetic differentiation among ponds. Within the pond network, we identified 3 to 4 genetic clusters. Combined with the detection of migrants, the results suggest an increase in gene flow within the restored pond network and that a high number of migrants came from the reference ponds. Our findings indicate an unexpected high dispersal ability for this small-bodied species. Overall, the absence of population structure represents a positive beginning for the restoration project. It also emphasizes the importance of spatial design in restoring a pond network and that such genetic data and methods should be used to monitor amphibians in restored habitats.     

Occurrence of Listeria spp. in Effluents of French Urban Wastewater Treatment Plants

Listeria spp. were found in most treated waters (84.4%) and raw sludge (89.2%) of six French urban wastewater treatment plants and one composting facility, examined monthly over a 1-year period. Most strains belonged to Listeria monocytogenes, serotypes 4b/4e being predominant. Sludge composting and liming reduced or prevented Listeria contamination.     

Galectin-7 in the control of epidermal homeostasis after injury

Galectins, a family of β-galactoside binding lectins, have recently emerged as novel regulators of tissue homeostasis. Galectin-7 is predominantly expressed in stratified epithelia, especially in epidermis. We report here the generation of galectin-7–deficient mice that are viable and do not display phenotypical abnormalities in skin structure or expression of epidermal markers. However, these mice show unique defects in the maintenance of epidermal homeostasis in response to environmental challenges. First, after UVB irradiation in vivo, the apoptotic response is prematurely triggered and lasts longer in the mutant epidermis. This result contrasts with the proapoptotic role that had been proposed for galectin-7. Second, wound-healing experiments in vivo revealed that galectin-7–deficient mice displayed a reduced reepithelialization potential compared with wild-type littermates. This effect could be attributed to a defect in cell migration. Because galectin-7 is located in the podosomes of keratinocytes migrating out of skin explants in culture, we propose that this glycan-binding protein may directly influence cell/extracellular matrix interactions. Finally, we also detected an unexpected intense hyperproliferative reaction consecutive to both types of stress in galectin-7–deficient mice. Together, these studies provide the first genetic evidence showing that galectin-7 can modulate keratinocyte apoptosis, proliferation, and migration during skin repair.     

Minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE)

One purpose of the biomedical literature is to report results in sufficient detail that the methods of data collection and analysis can be independently replicated and verified. Here we present reporting guidelines for gene expression localization experiments: the minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE). MISFISHIE is modeled after the Minimum Information About a Microarray Experiment (MIAME) specification for microarray experiments. Both guidelines define what information should be reported without dictating a format for encoding that information. MISFISHIE describes six types of information to be provided for each experiment: experimental design, biomaterials and treatments, reporters, staining, imaging data and image characterizations. This specification has benefited the consortium within which it was developed and is expected to benefit the wider research community. We welcome feedback from the scientific community to help improve our proposal.