Non-Canonical Wnt Predominates in Activated Rat Hepatic Stellate Cells,Influencing HSC Survival and Paracrine Stimulation of Kupffer Cells

The Wnt system is highly complex and is comprised of canonical and non-canonical pathways leading to the activation of gene expression. Our aim was to examine changes in the expression of Wnt ligands and regulators during hepatic stellate cell (HSC) transdifferentiation and assess the relative contributions of the canonical and non-canonical Wnt pathways in fibrogenic activated HSC. The expression profile of Wnt ligands and regulators in HSC was not supportive for a major role for β-catenin-dependent canonical Wnt signalling, this verified by inability to induce Topflash reporter activity in HSC even when expressing a constitutive active β-catenin. We detected expression of Wnt5a in activated HSC which can signal via non-canonical mechanisms and showed evidence for non-canonical signalling in these cells involving phosphorylation of Dvl2 and pJNK. Stimulation of HSC or Kupffer cells with Wnt5a regulated HSC apoptosis and expression of TGF-β1 and MCP1 respectively. We were unable to confirm a role for β-catenin-dependent canonical Wnt in HSC and instead propose autocrine and paracrine functions for Wnts expressed by activated HSC via non-canonical pathways. The data warrant detailed investigation of Wnt5a in liver fibrosis.     

Vaccination with Human Papillomavirus Pseudovirus-Encapsidated Plasmids Targeted to Skin Using Microneedles

Human papilloma virus-like particles (HPV VLP) serve as the basis of the current licensed vaccines for HPV. We have previously shown that encapsidation of DNA expressing the model antigen M/M2 from respiratory syncytial virus (RSV) in HPV pseudovirions (PsV) is immunogenic when delivered intravaginally. Because the HPV capsids confer tropism for basal epithelium, they represent attractive carriers for vaccination targeted to the skin using microneedles. In this study we asked: 1) whether HPV16 VLP administered by microneedles could induce protective immune responses to HPV16 and 2) whether HPV16 PsV-encapsidated plasmids delivered by microneedles could elicit immune responses to both HPV and the antigen delivered by the transgene. Mice immunized with HPV16 VLP coated microneedles generated robust neutralizing antibody responses and were protected from HPV16 challenge. Microneedle arrays coated with HPV16-M/M2 or HPV16-F protein (genes of RSV) were then tested and dose-dependent HPV and F-specific antibody responses were detected post-immunization, and M/M2-specific T-cell responses were detected post RSV challenge, respectively. HPV16 PsV-F immunized mice were fully protected from challenge with HPV16 PsV and had reduced RSV viral load in lung and nose upon intranasal RSV challenge. In summary, HPV16 PsV-encapsidated DNA delivered by microneedles induced neutralizing antibody responses against HPV and primed for antibody and T-cell responses to RSV antigens encoded by the encapsidated plasmids. Although the immunogenicity of the DNA component was just above the dose response threshold, the HPV-specific immunity was robust. Taken together, these data suggest microneedle delivery of lyophilized HPV PsV could provide a practical, thermostable combined vaccine approach that could be developed for clinical evaluation.     

A Chemical and Enzymatic Approach to Study Site-Specific Sumoylation

A variety of cellular pathways are regulated by protein modifications with ubiquitin-family proteins. SUMO, the Small Ubiquitin-like MOdifier, is covalently attached to lysine on target proteins via a cascade reaction catalyzed by E1, E2, and E3 enzymes. A major barrier to understanding the diverse regulatory roles of SUMO has been a lack of suitable methods to identify protein sumoylation sites. Here we developed a mass-spectrometry (MS) based approach combining chemical and enzymatic modifications to identify sumoylation sites. We applied this method to analyze the auto-sumoylation of the E1 enzyme in vitro and compared it to the GG-remnant method using Smt3-I96R as a substrate. We further examined the effect of smt3-I96R mutation in vivo and performed a proteome-wide analysis of protein sumoylation sites in Saccharomyces cerevisiae. To validate these findings, we confirmed several sumoylation sites of Aos1 and Uba2 in vivo. Together, these results demonstrate that our chemical and enzymatic method for identifying protein sumoylation sites provides a useful tool and that a combination of methods allows a detailed analysis of protein sumoylation sites.     

The cortisol awakening response (CAR) in male children with autism spectrum disorder

Our ability to adapt to change is fundamental. The cortisol awakening response (CAR) is a sharp rise in cortisol 30 min after waking to help prepare an individual for ensuing stress. Children with autism spectrum disorder (ASD) often have difficulty adapting to change. Exploration of the CAR is warranted; yet, the few studies investigating it are inconclusive. The CAR was investigated in 94 pre-pubertal male children 8-to-12 years of age with ASD (46) and typical development (TD, 48). Salivary samples were collected over three diurnal cycles involving two morning samples: M1: Immediately upon Waking and M2: 30-min Post Waking (M2 − M1 = CAR). The magnitude of the CAR was measured by independent two sample t-tests, variability was measured using Levene's Test, the sequence of the CAR was analyzed by a linear mixed-effects model and proportion of children exhibiting a CAR by chi-square test of independence. There were no significant differences on the CAR between the groups based on magnitude (t(92) = − 0.14, p = 0.89, d = 0.04), variability (F(45,47) = 1.11, p = 0.72, η² = 0.11) or the sequence over three days (F(2,88) = 0.26, p = 0.77, η² = 0.01). No significant differences were shown in the proportion of children exhibiting a CAR across the groups based on child (χ²(1) = 0.02, p = 0.89) or adult criterion (χ²(1) = 1.82, p = 0.18). Despite group differences in the regulation and responsivity of cortisol, the CAR is indistinguishable between children with and without ASD. Inconsistencies across studies may be due to age, criterion used, and diagnostic distinctions.     

Transcriptional profiling of bovine intervertebral disc cells: implications for identification of normal and degenerate human intervertebral disc cell phenotypes

Introduction  

Nucleus pulposus (NP) cells have a phenotype similar to articular cartilage (AC) cells. However, the matrix of the NP is clearly different to that of AC suggesting that specific cell phenotypes exist. The aim of this study was to identify novel genes that could be used to distinguish bovine NP cells from AC and annulus fibrosus (AF) cells, and to further determine their expression in normal and degenerate human intervertebral disc (IVD) cells.     

Heteroatom-linked indanylpyrazines are corticotropin releasing factor type-1 receptor antagonists

Low nanomolar corticotropin releasing factor type-1 (CRF(1)) receptor antagonists containing unique indanylamines were identified from the heteroatom-linked pyrazine chemotype. The most potent indanylpyrazine had a K(i)=11+/-1 nM. The oxygen-linked pyrazinyl derivatives were prepared through a copper-catalyzed coupling of a pyridinone to a bromo- or iodopyrazine.     

Enteropathogenic Escherichia coli Tir is an SH2/3 ligand that recruits and activates tyrosine kinases required for pedestal formation

Enteropathogenic Escherichia coli (EPEC) cause intestinal inflammation, severe diarrhoea and mortality, particularly among children in developing nations. Upon attachment to intestinal epithelial cells, EPEC induces actin-filled membrane protrusions called 'pedestals' and disrupts microvilli to form attaching and effacing (A/E) lesions. EPEC also disrupts epithelial barrier function and causes colitis. Here we have investigated how virulence factors which orchestrate formation of actin pedestals interface with host tyrosine kinases. We show that Tec-family tyrosine kinases localize beneath EPEC and, with Abl-family kinases, comprise a set of redundant host kinases utilized by EPEC to form actin pedestals. We also show that Tir, a virulence factor required for pathogenesis, contains a polyproline region (PPR) that interacts with SH3 domains of redundant kinases, and a phosphorylation site (Y474) that interacts with kinase SH2 domains. These interactions are essential for pedestal formation, and mimic activation of kinases by cellular ligands. Our results suggest that a positive feedback loop exists in which initial phosphorylation of Tir on Y474 by tyrosine kinases causes recruitment of additional redundant kinases via PPR-SH3 interactions and PO(3)-Y474-SH2 interactions, which in turn phosphorylate other Tir molecules as well as proteins that catalyse formation of actin pedestals.     

How the Location of Superoxide Generation Influences the β-Cell Response to Nitric Oxide

Cytokines impair the function and decrease the viability of insulin-producing β-cells by a pathway that requires the expression of inducible nitric oxide synthase (iNOS) and generation of high levels of nitric oxide. In addition to nitric oxide, excessive formation of reactive oxygen species, such as superoxide and hydrogen peroxide, has been shown to cause β-cell damage. Although the reaction of nitric oxide with superoxide results in the formation of peroxynitrite, we have shown that β-cells do not have the capacity to produce this powerful oxidant in response to cytokines. When β-cells are forced to generate peroxynitrite using nitric oxide donors and superoxide-generating redox cycling agents, superoxide scavenges nitric oxide and prevents the inhibitory and destructive actions of nitric oxide on mitochondrial oxidative metabolism and β-cell viability. In this study, we show that the β-cell response to nitric oxide is regulated by the location of superoxide generation. Nitric oxide freely diffuses through cell membranes, and it reacts with superoxide produced within cells and in the extracellular space, generating peroxynitrite. However, only when it is produced within cells does superoxide attenuate nitric oxide-induced mitochondrial dysfunction, gene expression, and toxicity. These findings suggest that the location of radical generation and the site of radical reactions are key determinants in the functional response of β-cells to reactive oxygen species and reactive nitrogen species. Although nitric oxide is freely diffusible, its biological function can be controlled by the local generation of superoxide, such that when this reaction occurs within β-cells, superoxide protects β-cells by scavenging nitric oxide.