Assessing phototoxicity in live fluorescence imaging.

Are the answers to biological questions obtained via live fluorescence microscopy substantially affected by phototoxicity? Although a single set of standards for assessing phototoxicity cannot exist owing to the breadth of samples and experimental questions associated with biological imaging, we need quantitative, practical assessments and reporting standards to ensure that imaging has a minimal impact on observed biological processes and sample health. Here we discuss the problem of phototoxicity in biology and suggest guidelines to improve its reporting and assessment.

Biallelic HERC1 mutations in a syndromic form of overgrowth and intellectual disability.

We report two Colombian siblings affected by overgrowth, intellectual disability and facial dysmorphism. Exome (via NGS) and Sanger sequencing revealed that biallelic sequence variants in a novel gene (HERC1) might be related to the disease pathogenesis. These results provide useful data for future genotype-phenotype correlations and for a molecular diagnosis of overgrowth.

A novel TGM1 mutation, leading to multiple splicing rearrangements, is associated with autosomal recessive congenital ichthyosis.

Autosomal recessive congenital ichthyosis (ARCI) is a group of rare, clinically heterogeneous skin disorders that affect cornification. ARCI includes lamellar ichthyosis, congenital ichthyosiform erythroderma and harlequin ichthyosis. TGM1 mutations cause > 50% of ARCI cases in the USA. We report two siblings with ARCI. They were found to carry a novel aetiological TGM1 mutation, which leads to the synthesis of multiple abnormal transcripts. These molecules resulted from three independent mechanisms: intron retention, exon skipping and activation of expand cryptic splice sites. Taken together, our findings expand the known TGM1 mutation repertoire, and provide an insight into the molecular mechanisms leading to ARCI phenotypes. These results could be useful for genetic counselling and future potential genotype-phenotype correlations.

Speckle reduction using an artificial neural network algorithm.

This paper presents an algorithm for reducing speckle noise from optical coherence tomography (OCT) images using an artificial neural network (ANN) algorithm. The noise is modeled using Rayleigh distribution with a noise parameter, sigma, estimated by the ANN. The input to the ANN is a set of intensity and wavelet features computed from the image to be processed, and the output is an estimated sigma value. This is then used along with a numerical method to solve the inverse Rayleigh function to reduce the noise in the image. The algorithm is tested successfully on OCT images of Drosophila larvae. It is demonstrated that the signal-to-noise ratio and the contrast-to-noise ratio of the processed images are increased by the application of the ANN algorithm in comparison with the respective values of the original images.

Functional evidence implicating FOXL2 in non-syndromic premature ovarian failure and in the regulation of the transcription factor OSR2.

BACKGROUND: FOXL2 encodes a forkhead transcription factor whose mutations are responsible for the blepharophimosis-ptosis-epicanthus inversus syndrome (BPES), involving craniofacial/palpebral abnormalities often associated with premature ovarian failure (POF). RESULTS: We describe a FOXL2 variant (p.Gly187Asp) in a case of POF without BPES. The subcellular localisation of FOXL2-G187D was normal but its transactivation capacity tested on two reporter promoters, one of which should be relevant to the ovary, was significantly lower than that of normal FOXL2. However, FOXL2-G187D was able to activate strongly a reporter construct driven by the promoter of Osr2 (odd-skipped related 2 transcription factor), which we have suggested to be a crucial target of FOXL2 in the craniofacial region. This is compatible with the absence of BPES in our patient. CONCLUSIONS: Our data provide evidence in favour of the implication of FOXL2 variants in non-syndromic POF and confirm the regulatory interaction between FOXL2 and OSR2 whose perturbation might contribute to the palpebral abnormalities observed in BPES patients.

[Genetic analysis of premature ovarian failure: role of forkhead and TGF-beta genes]. / Analyse génétique des insuffisances ovariennes prématurées: implication des gènes forkhead et TGF-beta.

Premature ovarian failure is a common pathology affecting 1% of women. Although multiple etiologies have been described the majority of cases are idiopathic. Forkhead transcription factors as FOXL2 and FOXO3A are of particular interest in the research of genetic factors related with the pathology as they are present in diverse developmental pathways and ovarian physiology. Similarly, some TGF-beta factors (i.e. BMP 15 and GDF-9) have been demonstrated to play a key role in the regulation, at ovarian level, of female reproduction. In recent years numerous studies have been performed in order to elucidate the implication of these factors in the ovarian physiopathology. The aim of this manuscript is to describe some of these advances in the context of premature ovarian failure.

Blobs and curves: object-based colocalisation for plant cells.

Blobs and curves occur everywhere in plant bioimaging: from signals of fluorescence-labelled proteins, through cytoskeletal structures, nuclei staining and cell extensions such as root hairs. Here we look at the problem of colocalisation of blobs with blobs (protein-protein colocalisation) and blobs with curves (organelle-cytoskeleton colocalisation). This article demonstrates a clear quantitative alternative to pixel-based colocalisation methods and, using object-based methods, can quantify not only the level of colocalisation but also the distance between objects. Included in this report are computational algorithms, biological experiments and guidance for those looking to increase their use of computationally-based and quantified analysis of bioimages.

Three-dimensional reconstruction of the antennal lobe in Drosophila melanogaster.

We present the first three-dimensional map of the antennal lobe of Drosophila melanogaster, based on confocal microscopic analysis of glomeruli stained with the neuropil-specific monoclonal antibody nc82. The analysis of confocal stacks allowed us to identify glomeruli according to the criteria shape, size, position, and intensity of antibody labeling. Forty glomeruli were labeled by nc82, eight of which have not been described before. Three glomeruli previously shown exclusively by backfills were not discernible in nc82 stainings. We distinguish three classes of glomeruli: (1) "landmark" glomeruli that are constant in all four criteria mentioned above, (2) less well-demarcated glomeruli that deviate in a single criterion, and (3) poorly defined glomeruli that vary in more than one criterion. All class 2 and 3 glomeruli can be identified by comparison with landmark neighbors. To further aid identification, our model assigns glomeruli to five arrays, each of which is defined by a prominent landmark glomerulus. Six glomeruli consist of distinct, but contiguous structural units, termed "compartments." Glomerular variability observed occasionally between males and females is in the same range as between individuals of the same sex, suggesting the lack of a significant sexual dimorphism in the glomerular pattern. We compare the new model with a previous map and address its potential for mapping activity and expression patterns. An important goal of this work was to create three-dimensional reference models of the antennal lobe, which are accessible on-line.

Mouse models for identifying genes modulating fertility parameters.

Fertility can be defined as the natural capability of giving life. It is an important factor both for human medicine, where ~10% of the couples call for the services of assisted reproductive technologies, and for species of economic interest. In particular, in dairy cows, the recent years have seen a kind of competition between milk production and fertility, and genes improving fertility are now considered as parameters to be selected for. The study of fertility pathways is nevertheless made difficult by the strong impact of environmental factors on this parameter, as well as by the number of genes potentially involved (as shown by systematic transcriptome analysis studies in the recent years). One additional level of complexity is given by the fact that factors modulating fertility will probably be sex specific. The usage of mouse models has been one of the solutions exploited for tackling with these difficulties. Here, we review three different approaches using mice for identifying genes modulating fertility in mammals: gene invalidation, positional cloning and in vitro mutagenesis. These three approaches exploit specific characteristics of the mouse, such as the possibility of controlling precisely the environment, an excellent genetic characterization and the existence of genomic and molecular tools equalled only in humans. Many indications suggest that at least some of the results obtained in mice could be easily transposed to the species of interest.

Using curve-fitting of curvilinear features for assessing registration of clinical neuropathology with in vivo MRI.

Traditional neuropathological examination provides information about neurological disease or injury of a patient at a high-resolution level. Correlating this type of post mortem diagnosis with in vivo image data of the same patient acquired by non-invasive tomographic scans greatly complements the interpretation of any disease or injury. We present the validation of a registration method for correlating macroscopic pathological images with MR images of the same patient. This also allows for 3-D mapping of the distribution of pathological changes throughout the brain. As the validation deals with datasets of widely differing sampling, we propose a method using smooth curvilinear anatomical features in the brain which allows interpolation between wide-spaced samples. Curvilinear features are common anatomically, and if selected carefully have the potential to allow determination of the accuracy of co-registration across large areas of a volume of interest.