Ceramide synthase TLCD3B is a novel gene associated with human recessive retinal dystrophy.

PURPOSE: Previous studies suggest that ceramide is a proapoptotic lipid as high levels of ceramides can lead to apoptosis of neuronal cells, including photoreceptors. However, no pathogenic variant in ceramide synthases has been identified in human patients and knockout of various ceramide synthases in mice has not led to photoreceptor degeneration. METHODS: Exome sequencing was used to identify candidate disease genes in patients with vision loss as confirmed by standard evaluation methods, including electroretinography (ERG) and optical coherence tomography. The vision loss phenotype in mice was evaluated by ERG and histological analyses. RESULTS: Here we have identified four patients with cone-rod dystrophy or maculopathy from three families carrying pathogenic variants in TLCD3B. Consistent with the phenotype observed in patients, the Tlcd3bKO/KO mice exhibited a significant reduction of the cone photoreceptor light responses, thinning of the outer nuclear layer, and loss of cone photoreceptors across the retina. CONCLUSION: Our results provide a link between loss-of-function variants in a ceramide synthase gene and human retinal dystrophy. Establishment of the Tlcd3b knockout murine model, an in vivo photoreceptor cell degeneration model due to loss of a ceramide synthase, will provide a unique opportunity in probing the role of ceramide in survival and function of photoreceptor cells.

Molecular Screening of 43 Brazilian Families Diagnosed with Leber Congenital Amaurosis or Early-Onset Severe Retinal Dystrophy.

Leber congenital amaurosis (LCA) is a severe disease that leads to complete blindness in children, typically before the first year of life. Due to the clinical and genetic heterogeneity among LCA and other retinal diseases, providing patients with a molecular diagnosis is essential to assigning an accurate clinical diagnosis. Using our gene panel that targets 300 genes that are known to cause retinal disease, including 24 genes reported to cause LCA, we sequenced 43 unrelated probands with Brazilian ancestry. We identified 42 unique variants and were able to assign a molecular diagnosis to 30/43 (70%) Brazilian patients. Among these, 30 patients were initially diagnosed with LCA or a form of early-onset retinal dystrophy, 17 patients harbored mutations in LCA-associated genes, while 13 patients had mutations in genes that were reported to cause other diseases involving the retina.

Guide wire reconstruction and visualization in 3DRA using monoplane fluoroscopic imaging.

A method has been developed that, based on the guide wire position in monoplane fluoroscopic images, visualizes the approximate guide wire position in the three-dimensional (3-D) vasculature, that is obtained prior to the intervention with 3-D rotational X-ray angiography (3DRA). The method assumes the position of the guide wire in the fluoroscopic images is known. A two-dimensional feature image is determined from the 3DRA data. In this feature image, the guide wire position is determined in a two-step approach: a mincost algorithm is used to determine a suitable position for the guide wire, and subsequently a snake optimization technique is applied to move the guide wire to a better position. The resulting guide wire can then be visualized in 3-D in combination with the 3DRA dataset. The reconstruction accuracy of the method has been evaluated using a 3DRA image of a vascular phantom filled with contrast, and monoplane fluoroscopic images of the same phantom without contrast and with a guide wire inserted. The evaluation has been performed for different projection angles, and with different parameters for the method. The final result does not appear to be very sensitive to the parameters of the method. The average mean error of the estimated 3-D guide wire position is 1.5 mm, and the average tip distance is 2.3 mm. The effect of inaccurate C-arm geometry information is also investigated. Small errors in geometry information (up to 1 degrees) will slightly decrease the 3-D reconstruction accuracies, with an error of at most 1 mm. The feasibility of this approach on clinical data is demonstrated.

Endpoint localization in guide wire tracking during endovascular interventions.

RATIONALE AND OBJECTIVES: A method is presented to track guide wires during endovascular interventions under X-ray fluoroscopy. Accurate guide wire tracking can be used to improve guide wire visualization in the low quality fluoroscopic images, and to estimate the position of the guide wire in world coordinates for navigation purposes. MATERIALS AND METHODS: A two-step procedure is used to track the guide wire in subsequent frames. First, the position of the guide wire is obtained by fitting a spline to the image. Subsequently, the spline is iteratively moved toward the tip of the guide wire for accurate tip localization. For both steps, a feature image is used in which line-like structures are enhanced. The method is validated using a reference standard, obtained by manual tracings of three observers. RESULTS: The method is evaluated on 20 image sequences, 10 sequences with a J-tipped guide wire and 10 with a straight guide wire. The tracking success was 96% for J-tipped and 100% for straight guide wires, whereas accurate endpoint localization could be performed in 91.3% and 94.4% of the frames respectively, with a tip localization error of less than 1.5 mm. CONCLUSIONS: Accurate endpoint localization can be performed for both J-tipped and straight guide wires and therefore the presented tracking method can be used for navigation purposes.

Three-dimensional guide-wire reconstruction from biplane image sequences for integrated display in 3-D vasculature.

Using three-dimensional rotational X-ray angiography (3DRA), three-dimensional (3-D) information of the vasculature can be obtained prior to endovascular interventions. However, during interventions, the radiologist has to rely on fluoroscopy images to manipulate the guide wire. In order to take full advantage of the 3-D information from 3DRA data during endovascular interventions, a method is presented that yields an integrated display of the position of the guide wire and vasculature in 3-D. The method relies on an automated method that tracks the guide wire simultaneously in biplane fluoroscopy images. Based on the calibrated geometry of the C-arm, the 3-D guide-wire position is determined and visualized in the 3-D coordinate system of the vasculature. The method is evaluated in an intracranial anthropomorphic vascular phantom. The influence of the angle between projections, distortion correction of the projection images, and accuracy of geometry knowledge on the accuracy of 3-D guide-wire reconstruction from biplane images is determined. If the calibrated geometry information is used and the images are corrected for distortion, a mean distance to the reference standard of 0.42 mm and a tip distance of 0.65 mm is found, which means that accurate guide-wire reconstruction from biplane images can be performed.

Guide-wire tracking during endovascular interventions.

A method is presented to extract and track the position of a guide wire during endovascular interventions under X-ray fluoroscopy. The method can be used to improve guide-wire visualization in low-quality fluoroscopic images and to estimate the position of the guide wire in world coordinates. A two-step procedure is utilized to track the guide wire in subsequent frames. First, a rough estimate of the displacement is obtained using a template-matching procedure. Subsequently, the position of the guide wire is determined by fitting a spline to a feature image. The feature images that have been considered enhance line-like structures on: 1) the original images; 2) subtraction images; and 3) preprocessed images in which coherent structures are enhanced. In the optimization step, the influence of the scale at which the feature is calculated and the additional value of using directional information is investigated. The method is evaluated on 267 frames from ten clinical image sequences. Using the automatic method, the guide wire could be tracked in 96% of the frames, with a similar accuracy to three observers, although the position of the tip was estimated less accurately.

The sng2 mutant of Arabidopsis is defective in the gene encoding the serine carboxypeptidase-like protein sinapoylglucose:choline sinapoyltransferase.

Serine carboxypeptidase-like (SCPL) proteins have traditionally been assigned roles in the hydrolytic processing of proteins; however, several SCPL proteins have recently been identified as catalysts in transacylation reactions of plant secondary metabolism. The novel functions of these enzymes suggest a catalytic diversity for plant SCPL proteins that extends beyond simple hydrolysis reactions. Characterization of the Arabidopsis sng2 (sinapoylglucose accumulator 2) mutant has identified another SCPL protein involved in plant secondary metabolism. The sng2 mutant was isolated by screening seed extracts for altered levels of sinapate esters, a group of phenylpropanoid compounds found in Arabidopsis and some other members of the Brassicaceae. Homozygous sng2 seeds accumulate sinapoylglucose instead of sinapoylcholine, and have increased levels of choline and decreased activity of the enzyme sinapoylglucose:choline sinapoyltransferase (SCT). Cloning of the SNG2 gene by a combination of map-based and candidate gene approaches demonstrates that SCT is another member of the growing class of SCPL acyltransferases involved in plant secondary metabolism.

Cloning of the SNG1 gene of Arabidopsis reveals a role for a serine carboxypeptidase-like protein as an acyltransferase in secondary metabolism.

Serine carboxypeptidases contain a conserved catalytic triad of serine, histidine, and aspartic acid active-site residues. These enzymes cleave the peptide bond between the penultimate and C-terminal amino acid residues of their protein or peptide substrates. The Arabidopsis Genome Initiative has revealed that the Arabidopsis genome encodes numerous proteins with homology to serine carboxypeptidases. Although many of these proteins may be involved in protein turnover or processing, the role of virtually all of these serine carboxypeptidase-like (SCPL) proteins in plant metabolism is unknown. We previously identified an Arabidopsis mutant, sng1 (sinapoylglucose accumulator 1), that is defective in synthesis of sinapoylmalate, one of the major phenylpropanoid secondary metabolites accumulated by Arabidopsis and some other members of the Brassicaceae. We have cloned the gene that is defective in sng1 and have found that it encodes a SCPL protein. Expression of SNG1 in Escherichia coli demonstrates that it encodes sinapoylglucose:malate sinapoyltransferase, an enzyme that catalyzes a transesterification instead of functioning like a hydrolase, as do the other carboxypeptidases. This finding suggests that SCPL proteins have acquired novel functions in plant metabolism and provides an insight into the evolution of secondary metabolic pathways in plants.

Modified lignin in tobacco and poplar plants over-expressing the Arabidopsis gene encoding ferulate 5-hydroxylase.

Ferulate 5-hydroxylase (F5H) is a cytochrome P450-dependent monooxygenase that catalyses the hydroxylation of ferulic acid, coniferaldehyde and coniferyl alcohol in the pathways leading to sinapic acid and syringyl lignin biosynthesis. Earlier studies in Arabidopsis have demonstrated that F5H over-expression increases lignin syringyl monomer content and abolishes the tissue-specificity of its deposition. To determine whether this enzyme has a similar regulatory role in plants that undergo secondary growth, we over-expressed the F5H gene in tobacco and poplar. In tobacco, over-expression of F5H under the control of the cauliflower mosaic virus 35S promoter increased lignin syringyl monomer content in petioles, but had no detectable effect on lignification in stems. By contrast, when the cinnamate 4-hydroxylase (C4H) promoter was used to drive F5H expression, there was a significant increase in stem lignin syringyl monomer content. Yields of thioglycolic acid and Klason lignin in C4H-F5H lines were lower than in the wild-type, suggesting that F5H over-expression leads to a reduced deposition or an altered extractability of lignin in the transgenic plants. Histochemical analysis suggested that the novel lignin in C4H-F5H transgenic lines was altered in its content of hydroxycinnamyl aldehydes. Transgenic poplar trees carrying the C4H-F5H transgene also displayed enhanced lignin syringyl monomer content. Taken together, these data show that hydroxylation of guaiacyl-substituted lignin precursors controls lignin monomer composition in woody plants, and that F5H over-expression is a viable metabolic engineering strategy for modifying lignin biosynthesis in forest species.

Lignin monomer composition is determined by the expression of a cytochrome P450-dependent monooxygenase in Arabidopsis.

The phenylpropanoid pathway provides precursors for the biosynthesis of soluble secondary metabolites and lignin in plants. Ferulate-5-hydroxylase (F5H) catalyzes an irreversible hydroxylation step in this pathway that diverts ferulic acid away from guaiacyl lignin biosynthesis and toward sinapic acid and syringyl lignin. This fact led us to postulate that F5H was a potential regulatory step in the determination of lignin monomer composition. To test this hypothesis, we have used Arabidopsis to examine the impact of F5H overexpression. Arabidopsis is a useful model system in which to study lignification because in wild-type plants, guaiacyl and syringyl lignins are deposited in a tissue-specific fashion, while the F5H-deficient fah1 mutant accumulates only guaiacyl lignin. Here we show that ectopic overexpression of F5H in Arabidopsis abolishes tissue-specific lignin monomer accumulation. Surprisingly, overexpression of F5H under the control of the lignification-associated cinnamate-4-hydroxylase promoter, but not the commonly employed cauliflower mosaic virus 35S promoter, generates a lignin that is almost entirely comprised of syringylpropane units. These experiments demonstrate that modification of F5H expression may enable engineering of lignin monomer composition in agronomically important plant species.