A novel strategy for delivering Niemann-Pick type C2 proteins across the blood–brain barrier using the brain endothelial-specific AAV-BR1 virus

Treating central nervous system (CNS) diseases is complicated by the incapability of numerous therapeutics to cross the blood–brain barrier (BBB), mainly composed of brain endothelial cells (BECs). Genetically modifying BECs into protein factories that supply the CNS with recombinant proteins is a promising approach to overcome this hindrance, especially in genetic diseases, like Niemann Pick disease type C2 (NPC2), where both CNS and peripheral cells are affected. Here, we investigated the potential of the BEC-specific adeno-associated viral vector (AAV-BR1) encoding NPC2 for expression and secretion from primary BECs cultured in an in vitro BBB model with mixed glial cells, and in healthy BALB/c mice. Transduced primary BECs had significantly increased NPC2 gene expression and secreted NPC2 after viral transduction, which significantly reversed cholesterol deposition in NPC2 deficient fibroblasts. Mice receiving an intravenous injection with AAV-BR1-NCP2-eGFP were sacrificed 8 weeks later and examined for its biodistribution and transgene expression of eGFP and NPC2. AAV-BR1-NPC2-eGFP was distributed mainly to the brain and lightly to the heart and lung, but did not label other organs including the liver. eGFP expression was primarily found in BECs throughout the brain but occasionally also in neurons suggesting transport of the vector across the BBB, a phenomenon also confirmed in vitro. NPC2 gene expression was up-regulated in the brain, and recombinant NPC2 protein expression was observed in both transduced brain capillaries and neurons. Our findings show that AAV-BR1 transduction of BECs is possible and that it may denote a promising strategy for future treatment of NPC2.

     

Predicting the Vulnerability of Great Apes to Disease: The Role of Superspreaders and Their Potential Vaccination

Disease is a major concern for the conservation of great apes, and one that is likely to become increasingly relevant as deforestation and the rise of ecotourism bring humans and apes into ever closer proximity. Consequently, it is imperative that preventative measures are explored to ensure that future epidemics do not wipe out the remaining populations of these animals. In this paper, social network analysis was used to investigate vulnerability to disease in a population of wild orang-utans and a community of wild chimpanzees. Potential ‘superspreaders’ of disease - individuals with disproportionately central positions in the community or population - were identified, and the efficacy of vaccinating these individuals assessed using simulations. Three resident female orang-utans were identified as potential superspreaders, and females and unflanged males were predicted to be more influential in disease spread than flanged males. By contrast, no superspreaders were identified in the chimpanzee network, although males were significantly more central than females. In both species, simulating the vaccination of the most central individuals in the network caused a greater reduction in potential disease pathways than removing random individuals, but this effect was considerably more pronounced for orang-utans. This suggests that targeted vaccinations would have a greater impact on reducing disease spread among orang-utans than chimpanzees. Overall, these results have important implications for orang-utan and chimpanzee conservation and highlight the role that certain individuals may play in the spread of disease and its prevention by vaccination.     

The hypothalamic arcuate nucleus: a key site for mediating leptin's effects on glucose homeostasis and locomotor activity

Leptin is required for normal energy and glucose homeostasis. The hypothalamic arcuate nucleus (ARH) has been proposed as an important site of leptin action. To assess the physiological significance of leptin signaling in the ARH, we used mice homozygous for a FLPe-reactivatable, leptin receptor null allele (Lepr(neo/neo) mice). Similar to Lepr(db/db) mice, these mice are obese, hyperglycemic, hyperinsulinemic, infertile, and hypoactive. To selectively restore leptin signaling in the ARH, we generated an adeno-associated virus expressing FLPe-recombinase, which was delivered unilaterally into the hypothalamus using stereotaxic injections. We found that unilateral restoration of leptin signaling in the ARH of Lepr(neo/neo) mice leads to a modest decrease in body weight and food intake. In contrast, unilateral reactivation markedly improved hyperinsulinemia and normalized blood glucose levels and locomotor activity. These data demonstrate that leptin signaling in the ARH is sufficient for mediating leptin's effects on glucose homeostasis and locomotor activity.     

A quantitative proteomic approach using two-dimensional gel electrophoresis and isotope-coded affinity tag labeling for studying human 20S proteasome heterogeneity

Mammalian proteasomes are macromolecular complexes formed of a catalytic 20S core associated to two regulatory complexes. The 20S core complex consists of four stacked rings of seven alpha or beta subunits. Three beta subunits contain a catalytic site and can be replaced by three interferon gamma-inducible counterparts to form the immunoproteasome. Cells may constitutively possess a mixture of both 20S proteasome types leading to a heterogeneous proteasome population. Purified rat 20S proteasome has been separated in several chromatographic fractions indicating an even higher degree of complexity in 20S proteasome subunit composition. This complexity may arise from the presence of subunit isoforms, as previously detected in purified human erythrocyte 20S proteasome. In this study, we have used a quantitative proteomic approach based on two-dimensional gel electrophoresis and isotope-coded affinity tag (ICAT) labeling to quantify the variations in subunit composition, including subunit isoforms, of 20S proteasomes purified from different cells. The protocol has been adapted to the analysis of low quantities of 20S proteasome complexes. The strategy has then been validated using standard proteins and has been applied to the comparison of 20S proteasomes from erythrocytes and U937 cancer cells. The results obtained show that this approach represents a valuable tool for the study of 20S proteasome heterogeneity.     

Laccase-catalyzed polymerization of tyrosine-containing peptides

Laccase-catalyzed polymerization of tyrosine and tyrosine-containing peptides was studied in the presence and absence of ferulic acid (FA). Advanced spectroscopic methods such as MALDI-TOF MS, EPR, FTIR microscopy and HPLC-fluorescence, as well as more conventional analytical tools: oxygen consumption measurements and SDS/PAGE were used in the reaction mechanism studies. Laccase was found to oxidize tyrosine and tyrosine-containing peptides, with consequent polymerization of the compounds. The covalent linkage connecting the compounds was found to be an ether bond. Only small amounts of dityrosine bonds were detected in the polymers. When FA was added to the reaction mixtures, it was found to be incorporated into the polymer structure. Thus, in addition to homopolymers, different heteropolymers containing two or four FA residues were formed in the reactions.     

Impacts of fever on locust life-history traits: costs or benefits?

Fever, like other mechanisms for defence against pathogens, may have positive and negative consequences for host fitness. In ectotherms, fever can be attained through modified behavioural thermoregulation. Here we examine potential costs of behavioural fever by holding adult, gregarious desert locusts at elevated temperatures simulating a range of fever intensities. We found no effect of fever temperatures on primary fitness correlates of survival and fecundity. However, flight capacity and mate competition were reduced, although there was no relation between time spent at fever temperatures and magnitude of the response. While these effects could indicate a direct cost of fever, they are also consistent with a shift towards the solitaria phase state that, in a field context, could be considered an adaptive life-history response to limit the impact of disease. These conflicting interpretations highlight the importance of considering complex defence mechanisms and trade-offs in an appropriate ecological context.     

Affinity ligand selection from a library of small molecules: assay development, screening, and application

A facile and cost-effective process for screening synthetic libraries for an affinity ligand is described. A high throughput 96-well plate filtration method was designed to screen both discrete compounds and mixtures of compounds attached to a solid support. Human serum albumin (HSA) was used as a target protein to demonstrate the proof of concept. Detection and quantitation by fluorescence was accomplished with the use of fluorescamine to conjugate the protein in the filtrate. It is found that mixtures demonstrating low average binding reflect an overall lower hit rate of the components, whereas deconvolution of mixtures with high protein binding consistently provides a high hit rate. This differs from many of the previous experiences screening solid-phase mixtures in which high false positive rates are noted to occur. A total of 100K compounds were tested: 25K as discrete samples and 75K as mixtures. An overall hit rate of 8% was observed. Secondary screening of compounds measured specificity, recovery, and dynamic binding capacity. The effectiveness of the method is illustrated using an affinity column made with a representative lead compound. A similar purity was achieved in a single-step purification of HSA from serum as compared to that obtained by two steps of ion-exchange chromatography. The process for primary screening of a large number of compounds is simple, inexpensive, and applicable to any soluble target protein of known or unknown function from crude mixtures and may have additional utility as a generic chemical affinity tool for the functional characterization of novel proteins emerging from proteomics work.     

Female-specific regulation of cuticular hydrocarbon biosynthesis by dopamine in Drosophila melanogaster

The role of dopamine (DA) is investigated in cuticular hydrocarbon biosynthesis in Drosophila melanogaster with three different approaches: use of DA-deficient mutants (dopa decarboxylase temperature sensitive mutants reared at restrictive temperature, and rescued by dopamine ingestion or by pale mutants partially rescued by a tyrosine hydroxylase construction), pharmacological treatments (tyrosine hydroxylase inhibitors) and topical application on decapitated flies. We report that DA specifically regulates diene hydrocarbon biosynthesis, which is female specific. Our results suggest that DA acts in adult flies within the first hours of imaginal life and that DA production from the brain is crucial for this process. Thus, DA contributes to reproduction in D. melanogaster by acting during a critical period during development of young adults.     

Immobilization of a novel antibacterial agent on solid phase and subsequent isolation of EF-Tu

Screening of our compound collection identified PNU-92560, a 2-[1,3,4]thiadiazolo[3,2-a]pyrimidine-6-carboxamide, as a novel antibacterial agent. Extensive analogue development identified that the 2-position of the thiadiazole could be functionalized with a linker that would allow the compound to be attached to a solid support. The extreme insolubility of the analogues prevented the mechanism of action for these compounds to be determined utilizing traditional methodology. The solid-supported compounds were utilized as affinity columns to identify elongation factor Tu (EF-Tu) as a putative target for this class of compounds. The activity of the compounds in a metabolic labeling experiments and in translation assay supports the identity of the target for these compounds to be EF-Tu.