Heterogeneous responses of temperate-zone amphibian populations to climate change complicates conservation planning.

The pervasive and unabated nature of global amphibian declines suggests common demographic responses to a given driver, and quantification of major drivers and responses could inform broad-scale conservation actions. We explored the influence of climate on demographic parameters (i.e., changes in the probabilities of survival and recruitment) using 31 datasets from temperate zone amphibian populations (North America and Europe) with more than a decade of observations each. There was evidence for an influence of climate on population demographic rates, but the direction and magnitude of responses to climate drivers was highly variable among taxa and among populations within taxa. These results reveal that climate drivers interact with variation in life-history traits and population-specific attributes resulting in a diversity of responses. This heterogeneity complicates the identification of conservation 'rules of thumb' for these taxa, and supports the notion of local focus as the most effective approach to overcome global-scale conservation challenges.

A method to assess the lysosomal residence of proteins in cultured cells.

Analytical subcellular fractionation is playing an increasingly important role in proteomic studies to identify and validate components of cellular organelles. For lysosomes, definitive studies in this area have been restricted to rodent tissues due to technical constraints. Our goal was to design a quantitative assay that would allow clear demonstration of lysosomal localization in cultured human cells. We found that culturing HepG2 (human hepatocellular carcinoma) cells in progesterone-containing medium elicited an extensive shift in the buoyant density of lysosomes as measured by isopycnic centrifugation in sucrose density gradients. The density of other organelles remained essentially unchanged; thus, this shift represents a specific test for lysosomal localization. Progesterone treatment of a variety of other cultured cells also elicited a shift in lysosome density. This approach should represent a valuable tool for identification and validation of both luminal and membrane lysosomal proteins.

Genotype analysis of the NF1 gene in the French Canadians from the Québec population.

We genotyped 19 NF1 families from the French Canadians of the Québec population with six intragenic polymorphic markers including 2 RFLPs (EcoRI and RsaI) and 4 microsatellites (IVS26-2.3, IVS27AC28.4, IVS27AC33.1, and IVS38GT53.0). Genotype analysis indicated families 7610 and 7473 bear deletions. In Family 7610 the deletion removed the entire NF1 gene except exons 1 to 4b. The breakpoint of the deletion is located between exons 4a and 4b. The deletion 7473 was derived from the maternal chromosome and exons 1 to 5 were deleted. The breakpoint of the deletion is located between exons 7 and 13. Their phenotypes are reported. The allele frequencies of microsatellites IVS27AC28.4 and IVS38GT53.0 are compared to previously reported data from Caucasians, including Spanish and Italians. The difference is statistically significant (P < 0.0036) for marker IVS27AC28.4 between the Québec French Canadian and the Italian population.

Identification and characterization of four novel large deletions in the human neurofibromatosis type 1 (NF1) gene.

We studied 20 unrelated NF1 patients by Southern blots with seven cDNA probes and loss of heterozygosity (LOH) analysis with four intragenic microsatellites (IVS26-2.3, IVS27AC28.4, IVS27AC33.1, and IVS38GT53.0). Four novel large deletions (178, 184, 236, and 237) have been identified and characterized. The breakpoint of deletion 178 was located in between exons 23-2 and 27b and the sequences downstream of the breakpoint were deleted. For deletion 184, the breakpoint was in between exons 27b and 29, and the region upstream of the breakpoint was deleted. With deletion 236, the breakpoint was in between exons 14 and 18 and the region downstream of the breakpoint was deleted. The breakpoint of deletion 237 was in between exons 38 and 45 and the sequences upstream of the breakpoint were deleted. These deletions were distributed randomly across the NF1 gene and no deletion hot spot was found. Our study suggests that the combination of analyses of loss of heterozygosity, southern blotting and southern blot densitometry can be used as a powerful method to detect large deletions, especially when family record is not available or the patient is a sporadic case.

Novel features in the tRNA-like world of plant viral RNAs.

tRNA-like domains are found at the 3' end of genomic RNAs of several genera of plant viral RNAs. Three groups of tRNA mimics have been characterized on the basis of their aminoacylation identity (valine, histidine and tyrosine) for aminoacyl-tRNA synthetases. Folding of these domains deviates from the canonical tRNA cloverleaf. The closest sequence similarities with tRNA are those found in valine accepting structures from tymoviruses (e.g. TYMV). All the viral tRNA mimics present a pseudoknotted amino acid accepting stem, which confers special structural and functional characteristics. In this review emphasis is given to newly discovered tRNA-like structures (e.g. in furoviruses) and to recent advances in the understanding of their three-dimensional architecture, which mimics L-shaped tRNA. Identity determinants in tRNA-like domains for aminoacylation are described, and evidence for their functional expression, as in tRNAs, is given. Properties of engineered tRNA-like domains are discussed, and other functional mimicries with tRNA are described (e.g. interaction with elongation factors and tRNA maturation enzymes). A final section reviews the biological role of the tRNA-like domains in amplification of viral genomes. In this process, in which the mechanisms can vary in specificity and efficiency according to the viral genus, function can be dependent on the aminoacylation properties of the tRNA-like domains and/or on structural properties within or outside these domains.

Myocardial tracking, a new method to calculate ejection fraction with gated SPECT: validation with (201)Tl versus planar angiography.

UNLABELLED: Left ventricular ejection fraction (LVEF) and viability are essential variables for the prognosis of myocardial infarction and can be measured simultaneously by (201)Tl gated SPECT; however, most algorithms tend to underestimate LVEF. This study aimed to evaluate a new myocardial tracking algorithm, MyoTrack (MTK), for automatic LVEF calculation. METHODS: A rest/redistribution (20 min/4 h) (201)Tl gated SPECT protocol followed immediately by a (99m)Tc equilibrium radionuclide angiography (ERNA) was performed in 75 patients with history of myocardial infarction. Quality of myocardial uptake was evaluated from count statistics and automatic quantification of defect sizes and severities (CardioMatch). LVEFs were calculated both with Germano's quantitative gated SPECT (QGS) algorithm and with MTK. Briefly, the originality of this algorithm resides in the unique end-diastole segmentation, matching to a template and motion field tracking throughout the cardiac cycle. RESULTS: ERNA LVEF averaged 33% +/- 14%. QGS significantly underestimated this value at 20 min (30% +/- 13%, P < 0.001) and at 4 h (30% +/- 13%, P < 0.0001). By contrast, MTK did not miscalculate LVEF at 20 min (34% +/- 14%, probability value was not significant) though a similar underestimation occurred at 4 h (31% +/- 13%, P < 0.02). Individual differences between early and late gated SPECT values and differences between gated SPECT and ERNA values did not correlate with the extension of perfusion defects, count statistics, or heart rate. CONCLUSION: MTK algorithm accurately calculates LVEF on early/high-count images compared with ERNA [corrected], even in patients with severe perfusion defects, but tends to underestimate LVEF on delayed/low-contrast images, as other algorithms do.

Specific tyrosylation of the bulky tRNA-like structure of brome mosaic virus RNA relies solely on identity nucleotides present in its amino acid-accepting domain.

Residues specifying aminoacylation by yeast tyrosyl-tRNA synthetase (TyrRS) of the tRNA-like structure present at the 3'-end of brome mosaic virus (BMV) RNA were determined by the in vitro approach using phage T7 transcripts. They correspond to nucleotides equivalent to base-pair C1-G72 and discriminator base A73 in the amino acid-acceptor branch of the molecule. No functional equivalents of the tyrosine anticodon residues, shown to be weakly involved in tyrosine identity of canonical tRNA(Tyr), were found in the BMV tRNA-like structure. This indicates a behaviour of this large and intricate molecule reminiscent of that of a minihelix derived from an amino acid-acceptor branch. Furthermore, iodine footprinting experiments performed on a tyrosylable BMV RNA transcript of 196 nt complexed to yeast TyrRS indicate that the amino acid-acceptor branch of the viral RNA is protected against cleavages as well as a hairpin domain, which is possibly located perpendicularly to its accepting branch. This domain without the canonical anticodon loop or the tyrosine anticodon acts as an anchor for TyrRS interaction leading to a better efficiency of tyrosylation.

A novel mutation in the neurofibromatosis type 1 (NF1) gene promotes skipping of two exons by preventing exon definition.

Using a protein truncation assay, we have identified a new mutation in the neurofibromatosis type 1 (NF1) gene that causes a severe defect in NF1 pre-mRNA splicing. The mutation, which consists of a G to A transition at position +1 of the 5' splice site of exon 12a, is associated with the loss of both exons 11 and 12a in the NF1 mRNA. Through the use of in vivo and in vitro splicing assays, we show that the mutation inactivates the 5' splice site of exon 12a, and prevents the definition of exon 12a, a process that is normally required to stimulate the weak 3' splice site of exon 12a. Because the 5' splice site mutation weakens the interaction of splicing factors with the 3' splice site of exon 12a, we propose that exon 11/exon 12a splicing is also compromised, leading to the exclusion of both exons 11 and 12a. Our results provide in vivo support for the importance of the exon definition model during NF1 splicing, and suggest that the NF1 region containing exons 11 and 12a plays an important role in the activity of neurofibromin.

Major tyrosine identity determinants in Methanococcus jannaschii and Saccharomyces cerevisiae tRNA(Tyr) are conserved but expressed differently.

Using in vitro tRNA transcripts and minihelices it was shown that the tyrosine identity for tRNA charging by tyrosyl-tRNA synthetase (TyrRS) from the archaeon Methanococcus jannaschii is determined by six nucleotides: the discriminator base A73 and the first base-pair C1-G72 in the acceptor stem together with the anticodon triplet. The anticodon residues however, participate only weakly in identity determination, especially residues 35 and 36. The completeness of the aforementioned identity set was verified by its tranfer into several tRNAs which then become as efficiently tyrosylatable as the wild-type transcript from M. jannaschii. Temperature dependence experiments on both the structure and the tyrosylation properties of M. jannaschii and yeast tRNA(Tyr) transcripts show that the archaeal transcript has greater structural stability and enhanced aminoacylation behaviour than the yeast transcript. Tyrosine identity in M. jannaschii is compared to that in yeast, and the conservation of the major determinant in both organisms, namely the C1-G72 pair, gives additional support to the existence of a functional connection between archaeal and eukaryotic aminoacylation systems.

Assessment of ejection fraction with Tl-201 gated SPECT in myocardial infarction: Precision in a rest-redistribution study and accuracy versus planar angiography.

BACKGROUND: Viability and left ventricular ejection fraction (LVEF) are essential measures for the assessment of myocardial infarction (MI). These 2 variables may be evaluated simultaneously by means of thallium-201 gated single photon emission computed tomography (SPECT); however, the precision and accuracy of LVEF measurements with this isotope remain controversial, particularly in cases of extended perfusion defects and poor count densities. METHODS AND RESULTS: Fifty patients with a history of MI underwent a 20-minute rest and a 4-hour redistribution Tl-201 gated SPECT viability protocol, immediately followed by a technetium-99m planar equilibrium radionuclide angiography (ERNA). On gated SPECT images, various count statistics were calculated, and perfusion was automatically quantified by means of CardioMatch, which provided both the size and severity of MI defects. Rest and redistribution LVEFs were determined from gated SPECT with Germano's algorithm, whereas LVEFs were calculated from ERNA using the manufacturer's software. Mean LVEF values calculated with rest gated SPECT, redistribution gated SPECT, and planar ERNA were 30% +/- 13%, 30% +/- 13% and 33% +/- 13%, respectively. Significant differences between repeated gated SPECT LVEFs were not shown by means of the paired t test. Correlation coefficients were high between 20-minute and 4-hour scans (r = 0.89) and between gated SPECT and ERNA (r = 0.88 and r = 0.92 at 20 minutes and 4 hours, respectively). Additionally, close agreement between gated SPECT and ERNA was shown by means of the Bland-Altman plot, despite an underestimation of 3 units. Finally, neither the technical conditions (count density, heart rate, lung uptake, etc) nor the perfusion alteration (size, severity, redistribution) appeared to interfere with the precision and accuracy of gated SPECT LVEF measurement. CONCLUSION: Tl-201 gated SPECT is a precise method for assessing LVEF within the same patient at 4-hour intervals, even with a substantial count decay, and it gives accurate results compared with planar ERNA, even in the case of large perfusion defects.

Statistical analysis of normal and abnormal dissymmetry in volumetric medical images.

We present a general method to study the dissymmetry of anatomical structures such as those found in the human brain. Our method relies on the estimate of 3D dissymmetry fields, the use of 3D vector field operators, and T2 statistics to compute significance maps. We also present a fully automated implementation of this method which relies mainly on the intensive use of a 3D non-rigid inter-patient matching tool. Such a tool is applied successively between the images and their symmetric versions with respect to an arbitrary plane, both to realign the images with respect to the mid-plane of the subject and to compute a dense 3D dissymmetry map. Inter-patient matching is also used to fuse the data of a population of subjects. We then describe three main application fields: the study of the normal dissymmetry within a given population, the comparison of the dissymmetry between two populations, and the detection of the significant abnormal dissymmetries of a patient with respect to a reference population. Finally, we present preliminary results illustrating these three applications for the case of the human brain.

Identity of tRNA for yeast tyrosyl-tRNA synthetase: tyrosylation is more sensitive to identity nucleotides than to structural features.

The specific aminoacylation of tRNA by yeast tyrosyl-tRNA synthetase does not rely on the presence of modified residues in tRNA(Tyr), although such residues stabilize its structure. Thus, the major tyrosine identity determinants were searched by the in vitro approach using unmodified transcripts produced by T7 RNA polymerase. On the basis of the tyrosylation efficiency of tRNA variants, the strongest determinants are base pair C1-G72 and discriminator residue A73 (the 5'-phosphoryl group on C1, however, is unimportant for tyrosylation). The three anticodon bases G34, U35, and A36 contribute also to the tyrosine identity, but to a lesser extent, with G34 having the most pronounced effect. Mutation of the GUA tyrosine anticodon into a CAU methionine anticodon, however, leads to a loss of tyrosylation efficiency similar to that obtained after mutation of the C1-G72 or A73 determinants. Transplantation of the six determinants into four different tRNA frameworks and activity assays on heterologous Escherichia coli and Methanococcus jannaschii tRNA(Tyr) confirmed the completeness of the tyrosine set and the eukaryotic character of the C1-G72 base pair. On the other hand, it was found that tyrosine identity in yeast does not rely on fine architectural features of the tRNA, in particular the size and sequence of the D-loop. Noticeable, yeast TyrRS efficiently charges a variant of E. coli tRNA(Tyr) with a large extra-region provided its G1-C72 base pair is changed to a C1-G72 base pair. Finally, tyrosylation activity is compatible with a +1 shift of the anticodon in the 3'-direction but is strongly inhibited if this shift occurs in the opposite 5'-direction.

Automatic detection of hippocampal atrophy on magnetic resonance images.

An automatic method for identifying hippocampal atrophy on magnetic resonance (MR) images obtained from patients with clinical evidence of temporal lobe epilepsy (TLE) is described. The method is based on the analysis of image intensity differences between patients and controls within a volume of interest (VOI) centred on the hippocampus. The core of the method is a fully automatic signal intensity-based inter-subject image registration technique. In particular, a global affine registration to a reference image is performed, followed by a local affine registration within the VOI. A mask produced by manual segmentation of the mean hippocampus for 30 control subjects enabled investigations to be restricted to a specified region of the VOI approximately corresponding to the hippocampus. Normal variations of hippocampal signal intensity were computed from images obtained for the 30 control subjects. The manual method of hippocampal volumetry, currently an important component of the pre-surgical evaluation of patients with clinical evidence of medically intractable TLE, is used to determine the lower 1st percentile limits of normal hippocampal volume. Hippocampi with volumes below this limit are defined as atrophic. We investigated whether the automatic method can correctly distinguish between 15 patients with significant hippocampal atrophy according to absolute volumes and a further 14 controls. ROC curves enabled evaluation of sensitivity and specificity in respect of an intensity threshold. 100% specificity is required when determining suitability of patients for neurosurgery, resulting in levels of 50% and 70% sensitivity in detecting atrophy in the right and left hippocampus, respectively. We propose that the method can be developed as an automatic screening procedure.

Factors affecting and computation of myocardial perfusion reference images.

Many quantitative analysis methods for myocardial perfusion studies require as a central step a comparison with a 'normal' or average density distribution map or reference image. It has been recognized, however, that the normal distribution can be affected by patient attributes, including sex and weight or body habitus, and by acquisition attributes, including the choice of tracer and the position of the patient during imaging. Some authors have proposed separate reference images for the sexes and the tracer. This approach fails if a large number of binary attributes have to be considered, since one would need 2" reference images for each attribute. The problem is compounded when continuous attributes (e.g. age and weight) are included, especially if the approach is to average separate homogeneous groups for each attribute. We propose to create case-specific reference images for the interpretation of myocardial perfusion studies by creating a model based on the influence of each attribute. From a non-homogeneous population of normal cases, or cases presumed to be normal on the basis of the Diamond and Forrester stratification, the effect of patient and study attributes on the density distribution in the stress image and the density differences between rest and stress images were computed. The effects are computed by multi-linear regression, to account for cross-correlation. Significance is assigned on the basis of a partial Fisher test. The data are myocardial perfusion images matched in 3D to a template by an elastic transformation. Even though there was some cross-correlation in the data, we were able to show independent effects of sex, position (prone or supine), age, weight, tracer combination and stress method (exercise, persantine and adenosine). Taken as a whole, the multi-linear regression demonstrated a significant effect in 72% of the pixels within the myocardial volume. In addition, the distribution predicted by the model was equivalent to average images from homogeneous matched groups. In conclusion, our approach makes it possible to produce case-specific reference images without the need for multiple homogeneous large groups to produce averages for each possible patient or study attribute.

Deformation analysis to detect and quantify active lesions in three-dimensional medical image sequences.

Evaluating precisely the temporal variations of lesion volumes is very important for at least three types of practical applications: pharmaceutical trials, decision making for drug treatment or surgery, and patient follow-up. In this paper we present a volumetric analysis technique, combining precise rigid registration of three-dimensional (3-D) (volumetric) medical images, nonrigid deformation computation, and flow-field analysis. Our analysis technique has two outcomes: the detection of evolving lesions and the quantitative measurement of volume variations. The originality of our approach is that no precise segmentation of the lesion is needed but the approximative designation of a region of interest (ROI) which can be automated. We distinguish between tissue transformation (image intensity changes without deformation) and expansion or contraction effects reflecting a change of mass within the tissue. A real lesion is generally the combination of both effects. The method is tested with synthesized volumetric image sequences and applied, in a first attempt to quantify in vivo a mass effect, to the analysis of a real patient case with multiple sclerosis (MS).

The peculiar architectural framework of tRNASec is fully recognized by yeast AspRS.

The wild-type transcript of Escherichia coli tRNASec, characterized by a peculiar core architecture and a large variable region, was shown to be aspartylatable by yeast AspRS. Similar activities were found for tRNASec mutants with methionine, leucine, and tryptophan anticodons. The charging efficiency of these molecules was found comparable to that of a minihelix derived from tRNAAsp and is accounted for by the presence of the discriminator residue G73, which is a major aspartate identity determinant. Introducing the aspartate identity elements from the anticodon loop (G34, U35, C36, C38) into tRNASec transforms this molecule into an aspartate acceptor with kinetic properties identical to tRNAAsp. Expression of the aspartate identity set in tRNASec is independent of the size of its variable region. The functional study was completed by footprinting experiments with four different nucleases as structural probes. Protection patterns by AspRS of transplanted tRNASec and tRNAAsp were found similar. They are modified, particularly in the anticodon loop, upon changing the aspartate anticodon into that of methionine. Altogether, it appears that recognition of a tRNA by AspRS is more governed by the presence of the aspartate identity set than by the structural framework that carries this set.

Automatic 3-D segmentation of internal structures of the head in MR images using a combination of similarity and free-form transformations: Part I, Methodology and validation on normal subjects.

The study presented in this paper tests the hypothesis that the combination of a global similarity transformation and local free-form deformations can be used for the accurate segmentation of internal structures in MR images of the brain. To quantitatively evaluate our approach, the entire brain, the cerebellum, and the head of the caudate have been segmented manually by two raters on one of the volumes (the reference volume) and mapped back onto all the other volumes, using the computed transformations. The contours so obtained have been compared to contours drawn manually around the structures of interest in each individual brain. Manual delineation was performed twice by the same two raters to test inter- and intrarater variability. For the brain and the cerebellum, results indicate that for each rater, contours obtained manually and contours obtained automatically by deforming his own atlas are virtually indistinguishable. Furthermore, contours obtained manually by one rater and contours obtained automatically by deforming this rater's own atlas are more similar than contours obtained manually by two raters. For the caudate, manual intra- and interrater similarity indexes remain slightly better than manual versus automatic indexes, mainly because of the spatial resolution of the images used in this study. Qualitative results also suggest that this method can be used for the segmentation of more complex structures, such as the hippocampus.

A novel and very peculiar HincII polymorphism in the 5' region of the human neurofibromatosis type 1 (NF1) gene.

We report a HincII polymorphism in the 5' end of the neurofibromatosis type 1 gene (NF1) as detected with a probe made of exons 1 to 4a (nucleotides 2 to 401 of the cDNA). This HincII site is most probably in an intron. Evidence presented suggests the probe reveals not one but two similar polymorphisms.

Image matching as a diffusion process: an analogy with Maxwell's demons.

In this paper, we present the concept of diffusing models to perform image-to-image matching. Having two images to match, the main idea is to consider the objects boundaries in one image as semi-permeable membranes and to let the other image, considered as a deformable grid model, diffuse through these interfaces, by the action of effectors situated within the membranes. We illustrate this concept by an analogy with Maxwell's demons. We show that this concept relates to more traditional ones, based on attraction, with an intermediate step being optical flow techniques. We use the concept of diffusing models to derive three different non-rigid matching algorithms, one using all the intensity levels in the static image, one using only contour points, and a last one operating on already segmented images. Finally, we present results with synthesized deformations and real medical images, with applications to heart motion tracking and three-dimensional inter-patients matching.