NPHP4 Variants Are Associated With Pleiotropic Heart Malformations

Rationale: Congenital heart malformations are a major cause of morbidity and mortality, especially in young children. Failure to establish normal left-right (L-R) asymmetry often results in cardiovascular malformations and other laterality defects of visceral organs. Objective: To identify genetic mutations causing cardiac laterality defects. Methods and Results: We performed a genome-wide linkage analysis in patients with cardiac laterality defects from a consanguineous family. The patients had combinations of defects that included dextrocardia, transposition of great arteries, double-outlet right ventricle, atrioventricular septal defects, and caval vein abnormalities. Sequencing of positional candidate genes identified mutations in NPHP4. We performed mutation analysis of NPHP4 in 146 unrelated patients with similar cardiac laterality defects. Forty-one percent of these patients also had laterality defects of the abdominal organs. We identified 8 additional missense variants that were absent or very rare in control subjects. To study the role of nphp4 in establishing L-R asymmetry, we used antisense morpholinos to knockdown nphp4 expression in zebrafish. Depletion of nphp4 disrupted L-R patterning as well as cardiac and gut laterality. Cardiac laterality defects were partially rescued by human NPHP4 mRNA, whereas mutant NPHP4 containing genetic variants found in patients failed to rescue. We show that nphp4 is involved in the formation of motile cilia in Kupffer's vesicle, which generate asymmetrical fluid flow necessary for normal L-R asymmetry. Conclusions: NPHP4 mutations are associated with cardiac laterality defects and heterotaxy. In zebrafish, nphp4 is essential for the development and function of Kupffer's vesicle cilia and is required for global L-R patterning

Loss of Niemann-Pick C1 or C2 Protein Results in Similar Biochemical Changes Suggesting That These Proteins Function in a Common Lysosomal Pathway

Niemann-Pick Type C (NPC) disease is a lysosomal storage disorder characterized by accumulation of unesterified cholesterol and other lipids in the endolysosomal system. NPC disease results from a defect in either of two distinct cholesterol-binding proteins: a transmembrane protein, NPC1, and a small soluble protein, NPC2. NPC1 and NPC2 are thought to function closely in the export of lysosomal cholesterol with both proteins binding cholesterol in vitro but they may have unrelated lysosomal roles. To investigate this possibility, we compared biochemical consequences of the loss of either protein. Analyses of lysosome-enriched subcellular fractions from brain and liver revealed similar decreases in buoyant densities of lysosomes from NPC1 or NPC2 deficient mice compared to controls. The subcellular distribution of both proteins was similar and paralleled a lysosomal marker. In liver, absence of either NPC1 or NPC2 resulted in similar alterations in the carbohydrate processing of the lysosomal protease, tripeptidyl peptidase I. These results highlight biochemical alterations in the lysosomal system of the NPC-mutant mice that appear secondary to lipid storage. In addition, the similarity in biochemical phenotypes resulting from either NPC1 or NPC2 deficiency supports models in which the function of these two proteins within lysosomes are linked closely

Calcium- and polyphosphate-containing acidic granules of sea urchin eggs are similar to acidocalcisomes, but are not the targets for NAADP

Acidocalcisomes are acidic calcium-storage compartments described from bacteria to humans and characterized by their high content in poly P (polyphosphate), a linear polymer of many tens to hundreds of Pi residues linked by high-energy phosphoanhydride bonds. In the present paper we report that millimolar levels of short-chain poly P (in terms of Pi residues) and inorganic PPi are present in sea urchin extracts as detected using 31P-NMR, enzymatic determinations and agarose gel electrophoresis. Poly P was localized to granules randomly distributed in the sea urchin eggs, as shown by labelling with the poly-P-binding domain of Escherichia coli exopolyphosphatase. These granules were enriched using iodixanol centrifugation and shown to be acidic and to contain poly P, as determined by Acridine Orange and DAPI (4′,6′-diamidino-2-phenylindole) staining respectively. These granules also contained large amounts of calcium, sodium, magnesium, potassium and zinc, as detected by X-ray microanalysis, and bafilomycin A1-sensitive ATPase, pyrophosphatase and exopolyphosphatase activities, as well as Ca2+/H+ and Na+/H+ exchange activities, being therefore similar to acidocalcisomes described in other organisms. Calcium release from these granules induced by nigericin was associated with poly P hydrolysis. Although NAADP (nicotinic acid–adenine dinucleotide phosphate) released calcium from the granule fraction, this activity was not significantly enriched as compared with the NAADP-stimulated calcium release from homogenates and was not accompanied by poly P hydrolysis. GPN (glycyl-L-phenylalanine-naphthylamide) released calcium when added to sea urchin homogenates, but was unable to release calcium from acidocalcisome-enriched fractions, suggesting that these acidic stores are not the targets for NAADP

Three-Dimensional Neurophenotyping of Adult Zebrafish Behavior

The use of adult zebrafish (Danio rerio) in neurobehavioral research is rapidly expanding. The present large-scale study applied the newest video-tracking and data-mining technologies to further examine zebrafish anxiety-like phenotypes. Here, we generated temporal and spatial three-dimensional (3D) reconstructions of zebrafish locomotion, globally assessed behavioral profiles evoked by several anxiogenic and anxiolytic manipulations, mapped individual endpoints to 3D reconstructions, and performed cluster analysis to reconfirm behavioral correlates of high- and low-anxiety states. The application of 3D swim path reconstructions consolidates behavioral data (while increasing data density) and provides a novel way to examine and represent zebrafish behavior. It also enables rapid optimization of video tracking settings to improve quantification of automated parameters, and suggests that spatiotemporal organization of zebrafish swimming activity can be affected by various experimental manipulations in a manner predicted by their anxiolytic or anxiogenic nature. Our approach markedly enhances the power of zebrafish behavioral analyses, providing innovative framework for high-throughput 3D phenotyping of adult zebrafish behavior

Fine-Scale Movements of the Broadnose Sevengill Shark and Its Main Prey, the Gummy Shark

Information on the fine-scale movement of predators and their prey is important to interpret foraging behaviours and activity patterns. An understanding of these behaviours will help determine predator-prey relationships and their effects on community dynamics. For instance understanding a predator's movement behaviour may alter pre determined expectations of prey behaviour, as almost any aspect of the prey's decisions from foraging to mating can be influenced by the risk of predation. Acoustic telemetry was used to study the fine-scale movement patterns of the Broadnose Sevengill shark Notorynchus cepedianus and its main prey, the Gummy shark Mustelus antarcticus, in a coastal bay of southeast Tasmania. Notorynchus cepedianus displayed distinct diel differences in activity patterns. During the day they stayed close to the substrate (sea floor) and were frequently inactive. At night, however, their swimming behaviour continually oscillated through the water column from the substrate to near surface. In contrast, M. antarcticus remained close to the substrate for the entire diel cycle, and showed similar movement patterns for day and night. For both species, the possibility that movement is related to foraging behaviour is discussed. For M. antarcticus, movement may possibly be linked to a diet of predominantly slow benthic prey. On several occasions, N. cepedianus carried out a sequence of burst speed events (increased rates of movement) that could be related to chasing prey. All burst speed events during the day were across the substrate, while at night these occurred in the water column. Overall, diel differences in water column use, along with the presence of oscillatory behaviour and burst speed events suggest that N. cepedianus are nocturnal foragers, but may opportunistically attack prey they happen to encounter during the day

Comparison of two tagging techniques for Sarpa salpa: external attachment and intraperitoneal. implantation

For a given species in a particular environment, the success and the harmlessness of transmitter attachment appear to be highly specific. As no information was available for the tagging of the strepie, Sarpa salpa, a suitability study of attachment techniques was needed. Two types of transmitter attachments were investigated: (1) externally, placed on the back of the fish below the dorsal fin by means of two threads sutured through the dorsal musculature, and (2) internally, in the peritoneal cavity. Twenty-five fish were monitored over a 14-d period for survival, surgical healing, injury occurrence and post-tagging behaviour. Even though we observed a 100% survival rate for both groups, with no noticeable buoyancy or swimming alteration, findings from this study indicate that both groups of tagged fish were affected by the presence of the transmitter in different ways. Fish with an external transmitter were chronically affected, incurring physical impact, whereas the surgically-implanted fish exhibited short-term tagging effects (less than 36 h), attributable to surgery effects, and were not subject to fouling on transmitter or wound chafing. Moreover, they appeared to recover quickly with no indication of long-term effects. Based on the findings of this study. the use of the surgical implantation method for Sarpa salpa is recommended.Pour une espèce donnée dans un environnement particulier, le succès d’attachement d’un émetteur s’avère hautement spécifique. Aucune information n’étant pour le moment disponible pour le marquage de la saupe, Sarpa salpa, un étude de faisabilité des techniques d’attachement d’émetteurs était nécessaire. Deux types de méthodes ont été testées : la première, externe, où l’émetteur est placé sur le dos du poisson en dessous de la nageoire dorsale à travers la musculature, et la seconde, interne, où l’émetteur est placé dans la cavité péritonéale. Vingt-cinq poissons ont été observés sur une période de deux semaines concernant le taux de survie, la cicatrisation post-chirurgicale, la présence ou non de blessures et le comportement post-opératoire. Même si nous avons observé un taux de survie de 100 % pour les deux groupes et n’avons pas observé de modification de la flottabilité ou de la nage, les conclusions de cette étude indiquent que les deux groupes de poissons ont été affectés par la présence de l’émetteur de manière différente. Les poissons avec une marque externe sont affectés de manière chronique, entraînant un impact physique. Quant aux poissons avec une marque dans la cavité péritonéale ils présentent des effets de courtes durées (moins de 36 h), qui peuvent être attribué à la chirurgie et ne sont pas sujet au fouling sur l’émetteur ni à une abrasion des blessures due au frottement du poisson. De plus, ils semblent récupérer plus rapidement, sans indication d’effets à long terme. Sur base des conclusions de cette étude nous recommandons l’utilisation de la méthode d’implantation intrapéritonéale pour marquer la saupe

Comparison of two tagging techniques for <i>Sarpa salpa</i>: external attachment and intraperitoneal implantation

For a given species in a particular environment, the success and the harmlessness of transmitter attachment appear to be highly specific. As no information was available for the tagging of the strepie, Sarpa salpa, a suitability study of attachment techniques was needed. Two types of transmitter attachments were investigated: (1) externally, placed on the back of the fish below the dorsal fin by means of two threads sutured through the dorsal musculature, and (2) internally, in the peritoneal cavity. Twenty-five fish were monitored over a 14-d period for survival, surgical healing, injury occurrence and post-tagging behaviour. Even though we observed a 100% survival rate for both groups, with no noticeable buoyancy or swimming alteration, findings from this study indicate that both groups of tagged fish were affected by the presence of the transmitter in different ways. Fish with an external transmitter were chronically affected, incurring physical impact, whereas the surgically-implanted fish exhibited short-term tagging effects (less than 36 h), attributable to surgery effects, and were not subject to fouling on transmitter or wound chafing. Moreover, they appeared to recover quickly with no indication of long-term effects. Based on the findings of this study. the use of the surgical implantation method for Sarpa salpa is recommended