A novel point mutation (L70P) inactivates poliovirus 3C protease.

Poliovirus (PV) contains a single-stranded positive-sense RNA genome, which is translated into a single polyprotein. Viral proteases process this polyprotein to produce several individual as well as fused proteins. The major viral protease 3C cleaves at nine of the eleven cleavage sites. During the process of expressing PV 3ABC protein in Escherichia coli, we identified a 3C mutant (L70P), which lost its protease activity. This loss of function was confirmed by generating recombinant adenoviruses expressing mutant and wild-type 3C. Further, infectious PV could not be recovered from PV full-length cDNA containing the L70P mutation. However, 3C L70P mutant cDNA could complement a PV cDNA containing a 1AB deletion, producing a viable virus population containing defective complementing genomes. Structural analysis of the mutant protein indicated that the L70P mutation resulted in the loss of a hydrogen bond between two residues located within a loop between two ß-sheets, potentially leading to strain on the catalytic site. We conclude that L70P inactivates 3C protease because of its close proximity to the 3C catalytic site.

Myelin genes are downregulated in canine fucosidosis.

The processes regulating the complex neurodegenerative cascade of vacuolation, neuroinflammation, neuronal loss and myelin deficits in fucosidosis, a neurological lysosomal storage disorder, remain unclear. To elucidate these processes the gene expression profile of the cerebral cortex from untreated and intrathecal enzyme replacement therapy treated fucosidosis pups and age-matched unaffected controls were examined. Neuroinflammation and cell death processes were identified to have a major role in fucosidosis pathophysiology with 37% of differentially expressed (DE) genes involved in these processes. Critical, specific, early decreases in expression levels of key genes in myelin assembly were identified by gene expression profiling, including myelin-associated glycoprotein (MAG), myelin and lymphocyte protein (MAL), and oligodendrocyte myelin paranodal and inner loop protein (OPALIN). These gene expression changes may be indicative of early neuronal loss causing reduced electrical impulses required for oligodendrocyte maturation.

Investigation of cerebrocortical and cerebellar pathology in canine fucosidosis and comparison to aged brain.

Fucosidosis is a fatal inherited neurodegenerative disease. The pathologic changes in brain which occur with progression from preclinical to late clinical disease were investigated in fucosidosis affected dogs. As aging also causes neurodegeneration and lysosomal dysfunction, pathologic markers of fucosidosis were compared to changes in the aging canine brain. Preclinical fucosidosis cerebral cortex and cerebellum revealed early increases in all neurodegenerative markers studied including apoptosis (2.1 fold), pyramidal neuronal loss (0.9 fold decrease) and Purkinje cell loss (1.2 fold decrease) compared to age matched controls. Increased axonal spheroid formation (>100 fold in cortex, 80 fold in cerebellum), microgliosis (9.2 fold) and astrocytosis (2.1 fold in cortex and 0.5 fold in cerebellum) were distinctive features of preclinical fucosidosis brain in all regions examined. This neuropathology progressed as the dogs developed severe clinical signs, with advanced fucosidosis brain exhibiting the greatest parenchymal destruction. These measures of the neurodegenerative and inflammatory changes in fucosidosis brain will assist monitoring disease progression and response to therapy.

The use of databases, data mining and immunoinformatics in vaccinology: where are we?

INTRODUCTION: Vaccinology has evolved from a sub-discipline focussed on simplistic vaccine development based on antibody-mediated protection to a separate discipline involving epidemiology, host and pathogen biology, immunology, genomics, proteomics, structure biology, protein engineering, chemical biology, and delivery systems. Data mining in combination with bioinformatics has provided a scaffold linking all these disciplines to the design of vaccines and vaccine adjuvants. Areas covered: This review provides background knowledge on immunological aspects which have been exploited with informatics for the in silico analysis of immune responses and the design of vaccine antigens. Furthermore, the article presents various databases and bioinformatics tools, and discusses B and T cell epitope predictions, antigen design, adjuvant research and systems immunology, highlighting some important examples, and challenges for the future. Expert opinion: Informatics and data mining have not only reduced the time required for experimental immunology, but also contributed to the identification and design of novel vaccine candidates and the determination of biomarkers and pathways of vaccine response. However, more experimental data is required for benchmarking immunoinformatic tools. Nevertheless, developments in immunoinformatics and reverse vaccinology, which are nascent fields, are likely to hasten vaccine discovery, although the path to regulatory approval is likely to remain a necessary impediment.

The use of BirA-BAP system to study the effect of US2 and US11 on MHC class I heavy chain in cells.

Biotinylation has been extensively used for antibody tagging, affinity-based purification, and in protein/DNA-protein interaction studies. Here we describe the use of biotinylation to study the turn-over of proteins in cells. We use the prokaryotic biotin ligase (BirA) to biotinylate the human leukocyte antigen (HLA)-A2 (A2) heavy chain (HC), which was engineered to contain a biotin acceptor peptide (BAP). Controlled availability of biotin in combination with visualization using streptavidin-conjugated peroxidase made it possible to detect biotinylated BAP-A2. Further, we exploited the effects of human cytomegalovirus (HCMV) unique short (US) proteins US2 and US11 on the turn-over of BAP-A2 HC. The full-length BAP-A2 HC and its mutants lacking either the cytosolic tail (tail-less) or both the transmembrane and cytosolic regions (soluble) were expressed via recombinant adenoviruses (rAd). The effect of US2, US11 and a control HCMV protein US9, also expressed via rAd, on each of the BAP- A2 forms was assessed. Experiments using this system showed that US2 and US11 cause proteasome-mediated degradation of full-length BAP-A2 HC but only US2 could cause degradation of tail-less BAP-A2. The results demonstrate that the technique of biotinylation can be used to study protein turn-over in cells.

Systems Pharmacogenomics Finds RUNX1 Is an Aspirin-Responsive Transcription Factor Linked to Cardiovascular Disease and Colon Cancer.

Aspirin prevents cardiovascular disease and colon cancer; however aspirin's inhibition of platelet COX-1 only partially explains its diverse effects. We previously identified an aspirin response signature (ARS) in blood consisting of 62 co-expressed transcripts that correlated with aspirin's effects on platelets and myocardial infarction (MI). Here we report that 60% of ARS transcripts are regulated by RUNX1 - a hematopoietic transcription factor - and 48% of ARS gene promoters contain a RUNX1 binding site. Megakaryocytic cells exposed to aspirin and its metabolite (salicylic acid, a weak COX-1 inhibitor) showed up regulation in the RUNX1 P1 isoform and MYL9, which is transcriptionally regulated by RUNX1. In human subjects, RUNX1 P1 expression in blood and RUNX1-regulated platelet proteins, including MYL9, were aspirin-responsive and associated with platelet function. In cardiovascular disease patients RUNX1 P1 expression was associated with death or MI. RUNX1 acts as a tumor suppressor gene in gastrointestinal malignancies. We show that RUNX1 P1 expression is associated with colon cancer free survival suggesting a role for RUNX1 in aspirin's protective effect in colon cancer. Our studies reveal an effect of aspirin on RUNX1 and gene expression that may additionally explain aspirin's effects in cardiovascular disease and cancer.

Oligodendrocyte loss during the disease course in a canine model of the lysosomal storage disease fucosidosis.

Hypomyelination is a poorly understood feature of many neurodegenerative lysosomal storage diseases, including fucosidosis in children and animals. To gain insight into hypomyelination in fucosidosis, we investigated lysosomal storage, oligodendrocyte death, and axonal and neuron loss in CNS tissues of fucosidosis-affected dogs aged 3 weeks to 42 months using immunohistochemistry, electron microscopy, and gene expression assays. Vacuole accumulation in fucosidosis oligodendrocytes commenced by 5 weeks of age; all oligodendrocytes were affected by 16 weeks. Despite progressive vacuolation, mature oligodendrocyte loss by apoptosis (caspase-6 positive) in the corpus callosum and cerebellar white matter stabilized by 16 weeks, with no further subsequent loss. Axonal neurofilament loss progressed only in late disease, suggesting that disturbed axon-oligodendrocyte interactions are unlikely to be the primary cause of hypomyelination. A 67% decline in the number of Purkinje cell layer oligodendrocytes coincided with a 67% increase in the number of caspase-6-positive Purkinje cells at 16 weeks, suggesting that early oligodendrocyte loss contributes to Purkinje cell apoptosis. Fucosidosis hypomyelination appeared to follow normal spatiotemporal patterns of myelination, with greater loss of oligodendrocytes and larger downregulation of CNP, MAL, and PLP1 genes at 16 weeks in the cerebellum versus the frontal cortex. These studies suggest that survival of oligodendrocytes in fucosidosis is limited during active myelination, although the mechanisms remain unknown.

Treatment of canine fucosidosis by intracisternal enzyme infusion.

The blood brain barrier is the major obstacle to treating lysosomal storage disorders of the central nervous system such as canine fucosidosis. This barrier was overcome by three, monthly injections of recombinant canine α-l-fucosidase enzyme were given intracisternally. In dogs treated from 8 weeks of age enzyme reached all areas of central nervous system as well as the cervical lymph node, bone marrow and liver. Brainstem and spinal cord samples from regions adjacent to the injection site had highest enzyme levels (39-73% of normal). Substantial enzyme activity (8.5-20% of normal controls) was found in the superficial brain compared to deeper regions (2.6-5.5% of normal). Treatment significantly reduced the fucosyl-linked oligosaccharide accumulation in most areas of CNS, liver and lymph node. In the surface and deep areas of lumbar spinal cord, oligosaccharide accumulation was corrected (79-80% reduction) to near normal levels (p<0.05). In the spinal meninges (thoracic and lumbar) enzyme activity (35-39% of normal control) and substrate reduction (58-63% affected vehicle treated samples) reached levels similar to those seen in phenotypically normal carriers (p<0.05).The procedure was safe and well-tolerated, treated (average 16%) dogs gained more weight (p<0.05) and there was no antibody formation or inflammatory reaction in plasma and CSF following treatments. The capacity of early ERT to modify progression of biochemical storage in fucosidosis is promising as this disease is currently only amenable to treatment by bone marrow transplantation which entails unacceptably high risks for many patients.