3D Printed Sugar-Sensing Hydrogels.

The ability of boronic acids (BAs) to reversibly bind diols, such as sugars, has been widely studied in recent years. In solution, through the incorporation of additional fluorophores, the BA-sugar interaction can be monitored by changes in fluorescence. Ultimately, a practical realization of this technology requires a transition from solution-based methodologies. Herein, the first example of 3D-printed sugar-sensing hydrogels, achieved through the incorporation of a BA-fluorophore pair in a gelatin methacrylamide-based matrix is presented. Through optimization of monomeric cocktails, it is possible to use extrusion printing to generate structured porous hydrogels which show a measurable and reproducible linear fluorescence response to glucose and fructose up to 100 mm.

The serine protease HtrA1 contributes to the formation of an extracellular 25-kDa apolipoprotein E fragment that stimulates neuritogenesis.

Apolipoprotein-E (apoE) is a glycoprotein highly expressed in the brain, where it appears to play a role in lipid transport, ß-amyloid clearance, and neuronal signaling. ApoE proteolytic fragments are also present in the brain, but the enzymes responsible for apoE fragmentation are unknown, and the biological activity of specific apoE fragments remains to be determined. Here we utilized SK-N-SH neuroblastoma cells differentiated into neurons with all-trans-retinoic acid (ATRA) to study extracellular apoE proteolysis. ApoE fragments were detectable in culture supernatants after 3 days, and their levels were increased for up to 9 days in the presence of ATRA. The concentration of apoE fragments was positively correlated with levels of the neuronal maturation markers (PSD95 and SMI32). The most abundant apoE fragments were 25- and 28-kDa N-terminal fragments that both contained sialylated glycosylation and bound to heparin. We detected apoE fragments only in the extracellular milieu and not in cell lysates, suggesting that an extracellular protease contributes to apoE fragmentation. Of note, siRNA-mediated knockdown of high-temperature requirement serine peptidase A1 (HtrA1) and a specific HtrA1 inhibitor reduced apoE 25-kDa fragment formation by 41 and 86%, respectively. Recombinant 25-kDa fragment apoE and full-length apoE both stimulated neuritogenesis in vitro, increasing neuroblastoma neurite growth by more than 2-fold over a 6-day period. This study provides a cellular model for assessing apoE proteolysis, indicates that HtrA1 regulates apoE 25-kDa fragment formation under physiological conditions, and reveals a new neurotrophic function for the apoE 25-kDa fragment.

Deletion of Abca7 increases cerebral amyloid-ß accumulation in the J20 mouse model of Alzheimer's disease.

ATP-binding cassette transporter A7 (ABCA7) is expressed in the brain and has been detected in macrophages, microglia, and neurons. ABCA7 promotes efflux of lipids from cells to apolipoproteins and can also regulate phagocytosis and modulate processing of amyloid precursor protein (APP) to generate the Alzheimer's disease (AD) amyloid-ß (Aß) peptide. Genome-wide association studies have indicated that ABCA7 single nucleotide polymorphisms confer increased risk for late-onset AD; however, the role that ABCA7 plays in the brain in the AD context is unknown. In the present study, we crossed ABCA7-deficient (A7(-/-)) mice with J20 amyloidogenic mice to address this issue. We show that ABCA7 loss doubled insoluble Aß levels and thioflavine-S-positive plaques in the brain. This was not related to changes in APP processing (assessed by analysis of full-length APP and the APP ß C-terminal fragment). Apolipoprotein E regulates cerebral Aß homeostasis and plaque load; however, the apolipoprotein E concentration was not altered by ABCA7 loss. Spatial reference memory was significantly impaired in both J20 and J20/A7(-/-) mice compared with wild-type mice; however, there were no cognitive differences between J20 and J20/A7(-/-) mice. There were also no major differences detected in hippocampal or plaque-associated microglial/macrophage markers between J20 and J20/A7(-/-) mice, whereas the capacity for bone marrow-derived macrophages derived from A7(-/-) mice to take up oligomeric Aß was reduced by 51% compared with wild-type mice. Our results suggest that ABCA7 plays a role in the regulation of Aß homeostasis in the brain and that this may be related to altered phagocyte function.

3D hybrid printing platform for auricular cartilage reconstruction.

As scaffolds approach dimensions that are of clinical relevance, mechanical integrity and distribution becomes an important factor to the overall success of the implant. Hydrogels often lack the structural integrity and mechanical properties for use in vivo or handling. The inclusion of a structural support during the printing process, referred to as hybrid printing, allows the implant to retain structure and protect cells during maturation without needing to compromise its biological performance. In this study, scaffolds for the purpose of auricular cartilage reconstruction were evaluated via a hybrid printing approach using methacrylated Gelatin (GelMA) and Hyaluronic acid (HAMA) as the cell-laden hydrogel, Polycaprolactone (PCL) as structural support and Lutrol F-127 as sacrificial material. Furthermore, printing parameters such as nozzle diameter, strand spacing and filament orientation scaffolds were investigated. Compression and bending tests showed that increasing nozzle sizes decrease the compressive modulus of printed scaffolds, with up to 82% decrease in modulus when comparing between a 400 µm and 200 µm sized nozzle tip at the same strand spacing. On the contrary, strand spacing and orientation influences mainly the bending modulus due to the greater porosity and changes in pore size area. Using a 400 µm sized nozzle, scaffolds fabricated have a measured compression and bending modulus in the range similar to the native cartilage. The viability and proliferation of human mesenchymal stem cells delivered within the bioink was not affected by the printing process. Using results obtained from mechanical testing, a scaffold with matching mechanical properties across six distinct regions mimicking the human auricular cartilage can be completed in one single print process. The use of PCL and GelMA-HAMA as structural support and cell-laden hydrogel respectively are an excellent combination to provide tailored mechanical integrity, while maintaining porosity and protection to cells during differentiation.

Cell Type-Specific Modulation of Cobalamin Uptake by Bovine Serum.

Tracking cellular 57Co-labelled cobalamin (57Co-Cbl) uptake is a well-established method for studying Cbl homeostasis. Previous studies established that bovine serum is not generally permissive for cellular Cbl uptake when used as a supplement in cell culture medium, whereas supplementation with human serum promotes cellular Cbl uptake. The underlying reasons for these differences are not fully defined. In the current study we address this question. We extend earlier observations by showing that fetal calf serum inhibits cellular 57Co-Cbl uptake by HT1080 cells (a fibrosarcoma-derived fibroblast cell line). Furthermore, we discovered that a simple heat-treatment protocol (95°C for 10 min) ameliorates this inhibitory activity for HT1080 cell 57Co-Cbl uptake. We provide evidence that the very high level of haptocorrin in bovine serum (as compared to human serum) is responsible for this inhibitory activity. We suggest that bovine haptocorrin competes with cell-derived transcobalamin for Cbl binding, and that cellular Cbl uptake may be minimised in the presence of large amounts of bovine haptocorrin that are present under routine in vitro cell culture conditions. In experiments conducted with AG01518 cells (a neonatal foreskin-derived fibroblast cell line), overall cellular 57Co-Cbl uptake was 86% lower than for HT1080 cells, cellular TC production was below levels detectable by western blotting, and heat treatment of fetal calf serum resulted in only a modest increase in cellular 57Co-Cbl uptake. We recommend a careful assessment of cell culture protocols should be conducted in order to determine the potential benefits that heat-treated bovine serum may provide for in vitro studies of mammalian cell lines.

Cerebral Apolipoprotein-D Is Hypoglycosylated Compared to Peripheral Tissues and Is Variably Expressed in Mouse and Human Brain Regions.

Recent studies have shown that cerebral apoD levels increase with age and in Alzheimer's disease (AD). In addition, loss of cerebral apoD in the mouse increases sensitivity to lipid peroxidation and accelerates AD pathology. Very little data are available, however, regarding the expression of apoD protein levels in different brain regions. This is important as both brain lipid peroxidation and neurodegeneration occur in a region-specific manner. Here we addressed this using western blotting of seven different regions (olfactory bulb, hippocampus, frontal cortex, striatum, cerebellum, thalamus and brain stem) of the mouse brain. Our data indicate that compared to most brain regions, the hippocampus is deficient in apoD. In comparison to other major organs and tissues (liver, spleen, kidney, adrenal gland, heart and skeletal muscle), brain apoD was approximately 10-fold higher (corrected for total protein levels). Our analysis also revealed that brain apoD was present at a lower apparent molecular weight than tissue and plasma apoD. Utilising peptide N-glycosidase-F and neuraminidase to remove N-glycans and sialic acids, respectively, we found that N-glycan composition (but not sialylation alone) were responsible for this reduction in molecular weight. We extended the studies to an analysis of human brain regions (hippocampus, frontal cortex, temporal cortex and cerebellum) where we found that the hippocampus had the lowest levels of apoD. We also confirmed that human brain apoD was present at a lower molecular weight than in plasma. In conclusion, we demonstrate apoD protein levels are variable across different brain regions, that apoD levels are much higher in the brain compared to other tissues and organs, and that cerebral apoD has a lower molecular weight than peripheral apoD; a phenomenon that is due to the N-glycan content of the protein.

Impaired lysosomal cobalamin transport in Alzheimer's disease.

Cobalamin (vitamin B12) is required for erythrocyte formation and DNA synthesis and it plays a crucial role in maintaining neurological function. As a coenzyme for methionine synthase and methylmalonyl-CoA mutase, cobalamin utilization depends on its efficient transit through the intracellular lysosomal compartment and subsequent delivery to the cytosol and mitochondria. Lysosomal function deteriorates in Alzheimer's disease (AD). Lysosomal acidification is defective in AD and lysosomal proteolysis is disrupted by AD-related presenilin 1 mutation. In this study, we propose that AD related lysosomal dysfunction may impair lysosomal cobalamin transport. The experiments use in vitro and in vivo models of AD to define how lysosomal dysfunction directly affects cobalamin utilization. SH-SY5Y-AßPP mutant cells were treated with a proteasome inhibitor to induce lysosomal amyloid-ß accumulation. We metabolically labeled these cells with [57Co] cobalamin and isolated purified lysosomes, mitochondria, and cytosol fractions. The results indicated that proteasome inhibition was associated with lysosomal amyloid-ß accumulation and a doubling of lysosomal [57Co] cobalamin levels. We also used AßPPxPS1 transgenic AD mice that were intraperitoneally injected with [57Co] cobalamin. The amount of [57Co] cobalamin in the major organs of these mice was measured and the subcellular [57Co] cobalamin distribution in the brain was assessed. The results demonstrated that lysosomal [57Co] cobalamin level was significantly increased by 56% in the AßPPxPS1 AD mouse brains as compared to wild type control mice. Together these data provide evidence that lysosomal cobalamin may be impaired in AD in association with amyloid-ß accumulation.

Apolipoprotein D modulates amyloid pathology in APP/PS1 Alzheimer's disease mice.

Apolipoprotein D (apoD) is expressed in the brain and levels are increased in affected brain regions in Alzheimer's disease (AD). The role that apoD may play in regulating AD pathology has not been addressed. Here, we crossed both apoD-null mice and Thy-1 human apoD transgenic mice with APP-PS1 amyloidogenic AD mice. Loss of apoD resulted in a nearly 2-fold increase in hippocampal amyloid plaque load, as assessed by immunohistochemical staining. Conversely, transgenic expression of neuronal apoD reduced hippocampal plaque load by approximately 35%. This latter finding was associated with a 60% decrease in amyloid ß 1-40 peptide levels, and a 34% decrease in insoluble amyloid ß 1-42 peptide. Assessment of ß-site amyloid precursor protein cleaving enzyme-1 (BACE1) levels and proteolytic products of amyloid precursor protein and neuregulin-1 point toward a possible association of altered BACE1 activity in association with altered apoD levels. In conclusion, the current studies provide clear evidence that apoD regulates amyloid plaque pathology in a mouse model of AD.

Perturbation of neuronal cobalamin transport by lysosomal enzyme inhibition.

Cbl (cobalamin) utilization as an enzyme cofactor is dependent on its efficient transit through lysosomes to the cytosol and mitochondria. We have previously proposed that pathophysiological perturbations in lysosomal function may inhibit intracellular Cbl transport with consequences for down-stream metabolic pathways. In the current study, we used both HT1080 fibroblasts and SH-SY5Y neurons to assess the impact that protease inhibitors, chloroquine and leupeptin (N-acetyl-L-leucyl-L-leucyl-L-argininal), have on the distribution of [57Co]Cbl in lysosomes, mitochondria and cytosol. Under standard cell culture conditions the distribution of [57Co]Cbl in both neurons and fibroblasts was ~5% in lysosomes, 14% in mitochondria and 81% in cytosol. Treatment of cells with either 25 µM chloroquine or 40 µM leupeptin for 48 h significantly increased the lysosomal [57Co]Cbl levels, by 4-fold in fibroblasts and 10-fold in neurons, and this was associated with reduced cytosolic and mitochondrial [57Co]Cbl concentrations. Based on Western blotting of LAMP2 in fractions recovered from an OptiPrep density gradient, lysosomal Cbl trapping was associated with an expansion of the lysosomal compartment and an increase in a subpopulation of lysosomes with increased size and density. Moreover, the decreased mitochondrial Cbl that was associated with lysosomal Cbl trapping was correlated with decreased incorporation of [14C] propionate into cellular proteins/macromolecules, indicating an inhibition of Cbl-dependent Mm-CoA (methylmalonyl-coenzyme A) mutase activity. These results add support to the idea that lysosomal dysfunction may significantly impact upon Cbl transport and utilization.

Analysis of subcellular [57Co] cobalamin distribution in SH-SY5Y neurons and brain tissue.

Cobalamin (Cbl) utilization as a cofactor for methionine synthase and methylmalonyl-CoA mutase is dependent on the transport of Cbl through lysosomes and its subsequent delivery to the cytosol and mitochondria. We speculated that neuropathological conditions that impair lysosomal function (e.g., age-related lipofuscinosis and specific neurodegenerative diseases) might impair lysosomal Cbl transport. To address this question, an appropriate method to quantify intracellular Cbl transport in neuronal cell types and brain tissue is required. Thus, we developed methods to measure [57Co] Cbl levels in lysosomes, mitochondria and cytosol obtained from in vitro and in vivo sources. Human SH-SY5Y neurons or HT1080 fibroblasts were labeled with [57Co] Cbl and homogenized using a ball-bearing homogenizer, and the lysates were separated into 10 fractions using ultracentrifugation in an OptiPrep density gradient. Lysosomes were recovered from the top of the gradient (fractions 1-5), which were clearly separated from mitochondria (fractions 7-9) on the basis of the expression of the marker proteins, LAMP2 and VDAC1. The isolated lysosomes were intact based on their colocalization with acid phosphatase activity. The lysosomal and mitochondrial fractions were free of the cytosolic markers beta-actin and methionine synthase. The relative distribution of [57Co] Cbl in both neurons and fibroblasts was as follows: 6% in the lysosomes, 14% in the mitochondria and 80% in the cytosol. This technique was also used to fractionate organelles from mouse brain, where marker proteins were detected in the gradient at positions similar to those observed for the cell lines, and the relative distribution of [57Co] Cbl was as follows: 12% in the lysosomes, 15% in the mitochondria and 73% in the cytosol. These methods provide a useful tool for the investigation of intracellular Cbl trafficking in a neurobiological setting.

Increased apolipoprotein D dimer formation in Alzheimer's disease hippocampus is associated with lipid conjugated diene levels.

Previous studies indicate that apolipoprotein D (apoD) may have a lipid antioxidant function in the brain. We have shown that apoD can reduce free radical-generating lipid hydroperoxides to inert lipid hydroxides in a reaction that involves conversion of surface exposed apoD methione-93 (Met93) residue to Met93-sulfoxide (Met93-SO). One consequence of this reaction is the formation of a stable dimerized form of apoD. As cerebral lipid peroxidation is associated with Alzheimer's disease (AD), in the present study we aimed to assess the possible presence of apoD dimers in postmortem hippocampal and cerebellar tissues derived from a cohort of pathologically defined cases ranging from control to late stage AD. Both soluble and insoluble (requiring guanidine HCl extraction) fractions of tissue homogenates were analyzed for apoD and its dimerized form. We also assessed amyloid-ß levels by ELISA and levels of lipid peroxidation by lipid conjugated diene and F2-isoprostane analysis. Our studies reveal a significant association between soluble apoD levels and AD Braak stage whereas apoD dimer formation appears to increase predominantly in the advanced stages of disease. The formation of apoD dimers is closely correlated to lipid conjugated diene levels and occurs in the hippocampus but not in the cerebellum. These results are consistent with the hypothesis that apoD acts as a lipid antioxidant in the brain.

Mitochondrial genome organization and phylogeny of two vespid wasps.

We sequenced the entire mitochondrial genome of Abispa ephippium (Hymenoptera: Vespoidea: Vespidae: Eumeninae) and most of the mitochondrial genome of Polistes humilis synoecus (Hymenoptera: Vespoidea: Vespidae: Polistinae). The arrangement of genes differed between the two genomes and also differed slightly from that inferred to be ancestral for the Hymenoptera. The genome organization for both vespids is different from that of all other mitochondrial genomes previously reported. A number of tRNA gene rearrangements were identified that represent potential synapomorphies for a subset of the Vespidae. Analysis of all available hymenopteran mitochondrial genome sequences recovered an uncontroversial phylogeny, one consistent with analyses of other types of data.

Mitochondrial genomes of Vanhornia eucnemidarum (Apocrita: Vanhorniidae) and Primeuchroeus spp. (Aculeata: Chrysididae): Evidence of rearranged mitochondrial genomes within the Apocrita (Insecta: Hymenoptera).

We sequenced most of the mitochondrial (mt) genomes of 2 apocritan taxa: Vanhornia eucnemidarum and Primeuchroeus spp. These mt genomes have similar nucleotide composition and codon usage to those of mt genomes reported for other Hymenoptera, with a total A + T content of 80.1% and 78.2%, respectively. Gene content corresponds to that of other metazoan mt genomes, but gene organization is not conserved. There are a total of 6 tRNA genes rearranged in V. eucnemidarum and 9 in Primeuchroeus spp. Additionally, several noncoding regions were found in the mt genome of V. eucnemidarum, as well as evidence of a sustained gene duplication involving 3 tRNA genes. We also report an inversion of the large and small ribosomal RNA genes in Primeuchroeus spp. mt genome. However, none of the rearrangements reported are phylogenetically informative with respect to the current taxon sample.

Domain-specific lipid distribution in macrophage plasma membranes.

Lipid rafts, defined as cholesterol- and sphingolipid-rich domains, provide specialized lipid environments understood to regulate the organization and function of many plasma membrane proteins. Growing evidence of their existence, protein cargo, and regulation is based largely on the study of isolated lipid rafts; however, the consistency and validity of common isolation methods is controversial. Here, we provide a detailed and direct comparison of the lipid and protein composition of plasma membrane "rafts" prepared from human macrophages by different methods, including several detergent-based isolations and a detergent-free method. We find that detergent-based and detergent-free methods can generate raft fractions with similar lipid contents and a biophysical structure close to that previously found on living cells, even in cells not expressing caveolin-1, such as primary human macrophages. However, important differences between isolation methods are demonstrated. Triton X-100-resistant rafts are less sensitive to cholesterol or sphingomyelin depletion than those prepared by detergent-free methods. Moreover, we show that detergent-based methods can scramble membrane lipids during the isolation process, reorganizing lipids previously in sonication-derived nonraft domains to generate new detergent-resistant rafts. The role of rafts in regulating the biological activities of macrophage plasma membrane proteins may require careful reevaluation using multiple isolation procedures, analyses of lipids, and microscopic techniques.