Juvenile CLN3 disease is a lysosomal cholesterol storage disorder: similarities with Niemann-Pick type C disease.

BACKGROUND: The most common form of neuronal ceroid lipofuscinosis (NCL) is juvenile CLN3 disease (JNCL), a currently incurable neurodegenerative disorder caused by mutations in the CLN3 gene. Based on our previous work and on the premise that CLN3 affects the trafficking of the cation-independent mannose-6 phosphate receptor and its ligand NPC2, we hypothesised that dysfunction of CLN3 leads to the aberrant accumulation of cholesterol in the late endosomes/lysosomes (LE/Lys) of JNCL patients' brains. METHODS: An immunopurification strategy was used to isolate intact LE/Lys from frozen autopsy brain samples. LE/Lys isolated from samples of JNCL patients were compared with age-matched unaffected controls and Niemann-Pick Type C (NPC) disease patients. Indeed, mutations in NPC1 or NPC2 result in the accumulation of cholesterol in LE/Lys of NPC disease samples, thus providing a positive control. The lipid and protein content of LE/Lys was then analysed using lipidomics and proteomics, respectively. FINDINGS: Lipid and protein profiles of LE/Lys isolated from JNCL patients were profoundly altered compared to controls. Importantly, cholesterol accumulated in LE/Lys of JNCL samples to a comparable extent than in NPC samples. Lipid profiles of LE/Lys were similar in JNCL and NPC patients, except for levels of bis(monoacylglycero)phosphate (BMP). Protein profiles detected in LE/Lys of JNCL and NPC patients appeared identical, except for levels of NPC1. INTERPRETATION: Our results support that JNCL is a lysosomal cholesterol storage disorder. Our findings also support that JNCL and NPC disease share pathogenic pathways leading to aberrant lysosomal accumulation of lipids and proteins, and thus suggest that the treatments available for NPC disease may be beneficial to JNCL patients. This work opens new avenues for further mechanistic studies in model systems of JNCL and possible therapeutic interventions for this disorder. FUNDING: San Francisco Foundation.

Lysosomal phospholipase A2 contributes to the biosynthesis of the atypical late endosome lipid bis(monoacylglycero)phosphate.

The late endosome/lysosome (LE/Lys) lipid bis(monoacylglycero)phosphate (BMP) plays major roles in cargo sorting and degradation, regulation of cholesterol and intercellular communication and has been linked to viral infection and neurodegeneration. Although BMP was initially described over fifty years ago, the enzymes regulating its synthesis remain unknown. The first step in the BMP biosynthetic pathway is the conversion of phosphatidylglycerol (PG) into lysophosphatidylglycerol (LPG) by a phospholipase A2 (PLA2) enzyme. Here we report that this enzyme is lysosomal PLA2 (LPLA2). We show that LPLA2 is sufficient to convert PG into LPG in vitro. We show that modulating LPLA2 levels regulates BMP levels in HeLa cells, and affects downstream pathways such as LE/Lys morphology and cholesterol levels. Finally, we show that in a model of Niemann-Pick disease type C, overexpressing LPLA2 alleviates the LE/Lys cholesterol accumulation phenotype. Altogether, we shed new light on BMP biosynthesis and contribute tools to regulate BMP-dependent pathways.

Proteomic fingerprinting of Neotropical hard tick species (Acari: Ixodidae) using a self-curated mass spectra reference library.

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry is an analytical method that detects macromolecules that can be used for proteomic fingerprinting and taxonomic identification in arthropods. The conventional MALDI approach uses fresh laboratory-reared arthropod specimens to build a reference mass spectra library with high-quality standards required to achieve reliable identification. However, this may not be possible to accomplish in some arthropod groups that are difficult to rear under laboratory conditions, or for which only alcohol preserved samples are available. Here, we generated MALDI mass spectra of highly abundant proteins from the legs of 18 Neotropical species of adult field-collected hard ticks, several of which had not been analyzed by mass spectrometry before. We then used their mass spectra as fingerprints to identify each tick species by applying machine learning and pattern recognition algorithms that combined unsupervised and supervised clustering approaches. Both Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) classification algorithms were able to identify spectra from different tick species, with LDA achieving the best performance when applied to field-collected specimens that did have an existing entry in a reference library of arthropod protein spectra. These findings contribute to the growing literature that ascertains mass spectrometry as a rapid and effective method to complement other well-established techniques for taxonomic identification of disease vectors, which is the first step to predict and manage arthropod-borne pathogens.

Asymmetry and bifurcations in three-dimensional sudden-contraction channel flows.

This study reports the presence of two different stable modes of bifurcation in the near field of a three-dimensional sudden contraction. To be precise, flow downstream of a symmetric sudden contraction undergoes a transition from a symmetric state to an asymmetric state through a symmetry-breaking pitchfork bifurcation following an increase in the channel aspect ratio or the Reynolds number. In addition, the oncoming (upstream) symmetry-plane flow exhibits spanwise bifurcations along the topological core lines of each of the salient roof and floor eddies. Small aspect-ratio (contraction) channels are noted to facilitate interesting splitting of the salient roof and floor eddies into multicore forms with accompanying spanwise flow bifurcations along the respective vortical core lines. Herein extensive three-dimensional simulations performed with various aspect and contraction ratios and Reynolds numbers clearly suggest that flow transition in the sudden-contraction channels should indeed occur primarily through these two generically distinct modes of bifurcation.

Surface self-organization caused by dislocation networks.

We report a new mechanism of self-organization that can lead to robust surface ordering. We have quantitatively analyzed the thermal motion of holes created by sulfur atoms in a silver monolayer on a ruthenium surface, which we observed in real time with scanning tunneling microscopy. We find that the stability of the array of holes is determined by the arrangement and structure of misfit dislocations in the film.

Three-dimensional simulation of square jets in cross-flow.

Direct numerical simulations are performed to predict the three-dimensional unsteady flow interactions in the near-field of a square jet issuing normal to a cross-flow. The simulated flow features reveal the formation of an upstream horseshoe vortex system, which is the result of an interaction between the oncoming channel floor shear layer and the transverse jet; the growth of a sequence of Kelvin-Helmholtz instability-induced vortical rollers in the mixing layer between the jet and the cross-flow, which wrap around the front side of the jet; and the inception process of the counter-rotating vortex pair (CVP), which is initiated through the folding of the lateral jet shear layers. It has been observed that for a square jet in cross-flow, the developed Kelvin-Helmholtz instability induced shear layer rollers do not form closed circumferential vortex rings. Along the downstream side of the jet, the extended tails of such rollers gradually join the locally evolving CVP. The very inception of the CVP is, however, observed to take place within the cross-flow-induced skewed lateral jet shear layers, and such inception was seen to occur slightly below the jet orifice. The simulated results also reveal the growth of the upright wake vortices from the topological singular points that developed on the cross-flow floor boundary layer. The accumulated floor vortices are seen to spiral around the critical points and subsequently leave the channel floor uprightly. During upward motion these vortices eventually get entrained into the CVP core. It has been made clear topologically that the unstable local surface excitations are the seeds from which upright vortices grow. Interestingly, such findings remain quite consistent with the existing experimental predictions for a round jet. Simulations were performed for two moderate values of the Reynolds number 225 and 300, based on the jet width and the average cross-flow inlet velocity, and for two different values of the jet to cross-stream velocity ratio, 2.5 and 3.5.

Interaction of trailing vortices in the wake of a wall-mounted rectangular cylinder.

Numerical simulations are performed to investigate three-dimensional unsteady vortex-vortex and vortex-surface interactions in the near field of a wall-mounted rectangular cylinder placed inside a channel. The generation mechanism of the upstream and the trailing vortices from the topologically important critical points and their near-wall evolution pattern have been examined in detail. In the upstream region, a laminar necklace vortex system formed around the junction between the rectangular block (cylinder) and the flat channel floor. A sequence of streamwise vortical rollers dominated the downstream interaction region, and they exhibited strong unsteady vortex-surface interaction. Streamwise vortices which formed upstream of the obstacle exhibited quadrupole structure with the dominant pair being central downwash, whereas those lifting the flow behind the obstacle were of predominantly central upwash. Notably, at some downstream location, the near-wall wake structure was observed to locally disappear due to mutual interaction and annihilation by opposite strength vortices on either side of the wake centerline. During the entire course of unsteady flow evolution, such a disappearance of the wake remained closely associated with local contraction of the limiting streamlines on the channel floor, the development of a pair of topologically important floor critical points (saddles), and the presence of a near-wall node on the vertical symmetry plane. The dominance of inward transverse flow toward these saddles together with flow evolution from the downstream node on the vertical symmetry plane were found to be particularly responsible for facilitating the local interaction of various vortices of opposite strength, leading to significant vorticity cancellation in the region. Moreover, the basic source of the wake vortices and their nature of evolution behind the cylinder were also investigated here, and they were found to be fundamentally different from what one usually observes in the near-wake of a transverse jet. However, the growth of a pair of vertically lifting vortices from the spiraling shear layer nodes just behind the downstream edge of the cylinder base was detected in this flow configuration also.

Spanwise bifurcation in plane-symmetric sudden-expansion flows.

Present computational investigation reports a steady bifurcation phenomenon for three-dimensional flows through a plane-symmetric sudden expansion. When the channel aspect ratio exceeds a critical value, the well-known step height (pitchfork) bifurcation evolves with different symmetry breaking orientations on the left and right sides of the channel and bifurcates in the spanwise direction. For the channel aspect ratio less than the critical value, the originally occurring spanwise bifurcation cannot be stably retained and evolves eventually to a step height bifurcation. Compared to step height bifurcation, the spanwise bifurcation is found to be more difficult to obtain, because the symmetric flow present on the spanwise symmetry plane is unstable in two dimensions. For completeness, an extensive analysis of the observed spanwise bifurcation, covering its transient behavior, dependence on flow Reynolds number, channel aspect ratio, and expansion ratio, is included.