Kupffer Cell Isolation for Nanoparticle Toxicity Testing

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle toxicity testing in immortalized cell lines or primary cells have shown discrepancies, highlighting the need to extend the use of primary cells for in vitro assays. This protocol describes the isolation of mouse liver macrophages, named Kupffer cells, and their use to study nanoparticle toxicity. Kupffer cells are the most abundant macrophage population in the body and constitute part of the reticulo-endothelial system (RES), responsible for the capture of circulating nanoparticles. The Kupffer cell isolation method reported here is based on a 2-step perfusion method followed by purification on density gradient. The method, based on collagenase digestion and density centrifugation, is adapted from the original protocol developed by Smedsrød et al. designed for rat liver cell isolation and provides high yield (up to 14 x 10⁶ cells per mouse) and high purity (>95%) of Kupffer cells. This isolation method does not require sophisticated or expensive equipment and therefore represents an ideal compromise between complexity and cell yield. The use of heavier mice (35-45 g) improves the yield of the isolation method but also facilitates remarkably the procedure of portal vein cannulation. The toxicity of functionalized carbon nanotubes f-CNTs was measured in this model by the modified LDH assay. This method assesses cell viability by measuring the lack of structural integrity of Kupffer cell membrane after incubation with f-CNTs. Toxicity induced by f-CNTs can be measured consistently using this assay, highlighting that isolated Kupffer cells are useful for nanoparticle toxicity testing. The overall understanding of nanotoxicology could benefit from such models, making the nanoparticle selection for clinical translation more efficient.     

Increase in Dickkopf-1 Serum Level in Recent Spondyloarthritis. Data from the DESIR Cohort

Objectives

To investigate DKK-1 and SOST serum levels among patients with recent inflammatory back pain (IBP) fulfilling ASAS criteria for SpA and associated factors.

Methods

The DESIR cohort is a prospective, multicenter French cohort of 708 patients with early IBP (duration >3 months and <3 years) suggestive of AxSpA. DKK-1 and SOST serum levels were assessed at baseline and were compared between the subgroup of patients fulfilling ASAS criteria for SpA (n = 486; 68.6%) and 80 healthy controls.

Results

Mean SOST serum levels were lower in ASAS+ patients than healthy controls (49.21 ± 25.9 vs. 87.8 ± 26 pmol/L; p<0.0001). In multivariate analysis, age (p = 5.4 10⁻⁹), CRP level (p<0.0001) and serum DKK-1 level (p = 0.001) were associated with SOST level. Mean DKK-1 serum levels were higher in axial SpA patients than controls (30.03 ± 15.5 vs. 11.6 ± 4.2 pmol/L; p<0.0001). In multivariate analysis, DKK-1 serum levels were associated with male gender (p = 0.03), CRP level (p = 0.006), SOST serum level (p = 0.002) and presence of sacroiliitis on radiography (p = 0.05). Genetic association testing of 10 SNPs encompassing the DKK-1 locus failed to demonstrate a significant contribution of genetics to control of DKK-1 serum levels.

Conclusions

DKK-1 serum levels were increased and SOST levels were decreased among a large cohort of patients with early axial SpA compared to healthy controls. DKK-1 serum levels were mostly associated with biological inflammation and SOST serum levels.     

Seeing More by Showing Less: Orientation-Dependent Transparency Rendering for Fiber Tractography Visualization

Fiber tractography plays an important role in exploring the architectural organization of fiber trajectories, both in fundamental neuroscience and in clinical applications. With the advent of diffusion MRI (dMRI) approaches that can also model “crossing fibers”, the complexity of the fiber network as reconstructed with tractography has increased tremendously. Many pathways interdigitate and overlap, which hampers an unequivocal 3D visualization of the network and impedes an efficient study of its organization. We propose a novel fiber tractography visualization approach that interactively and selectively adapts the transparency rendering of fiber trajectories as a function of their orientation to enhance the visibility of the spatial context. More specifically, pathways that are oriented (locally or globally) along a user-specified opacity axis can be made more transparent or opaque. This substantially improves the 3D visualization of the fiber network and the exploration of tissue configurations that would otherwise be largely covered by other pathways. We present examples of fiber bundle extraction and neurosurgical planning cases where the added benefit of our new visualization scheme is demonstrated over conventional fiber visualization approaches.     

New evidence for a 67,000-year-old human presence at Callao Cave, Luzon, Philippines

Documentation of early human migrations through Island Southeast Asia and Wallacea en route to Australia has always been problematic due to a lack of well-dated human skeletal remains. The best known modern humans are from Niah Cave in Borneo (40-42 ka), and from Tabon Cave on the island of Palawan, southwest Philippines (47 ± 11 ka). The discovery of Homo floresiensis on the island of Flores in eastern Indonesia has also highlighted the possibilities of identifying new hominin species on islands in the region. Here, we report the discovery of a human third metatarsal from Callao Cave in northern Luzon. Direct dating of the specimen using U-series ablation has provided a minimum age estimate of 66.7 ± 1 ka, making it the oldest known human fossil in the Philippines. Its morphological features, as well as size and shape characteristics, indicate that the Callao metatarsal definitely belongs to the genus Homo. Morphometric analysis of the Callao metatarsal indicates that it has a gracile structure, close to that observed in other small-bodied Homo sapiens. Interestingly, the Callao metatarsal also falls within the morphological and size ranges of Homo habilis and H. floresiensis. Identifying whether the metatarsal represents the earliest record of H. sapiens so far recorded anywhere east of Wallace’s Line requires further archaeological research, but its presence on the isolated island of Luzon over 65,000 years ago further demonstrates the abilities of humans to make open ocean crossings in the Late Pleistocene.     

Crystallization of the membrane protein hVDAC1 produced in cell-free system

Structural studies of membrane proteins are in constant evolution with the development of new improvements for their expression, purification, stabilization and crystallization. However, none of these methods still provides a universal approach to solve the structure of membrane proteins. Here we describe the crystallization of the human voltage-dependent anion channel-1 produced by a bacterial cell-free expression system. While VDAC structures have been recently solved, we propose an alternative strategy for producing the recombinant protein, which can be applied to other membrane proteins reluctant to expression, purification and crystallization by classical approaches. Despite a lot of efforts to crystallize a cell-free expressed membrane protein, this study is to our knowledge one of the first reports of a successful crystallization. Focusing on expression in a soluble and functional state, in a detergent environment, is the key to get crystals. Although the diffraction of VDAC crystals is limited, the simplicity and the rapidity to set-up and optimize this technology are drastic advantages in comparison to other methods.