Three-dimensional localization of pORF65 in Kaposi's sarcoma-associated herpesvirus capsid

Of the six herpesvirus capsid proteins, the smallest capsid proteins (SCPs) share the least sequence homology among herpesvirus family members and have been implicated in virus specificity during infection. The herpes simplex virus-1 (HSV-1) SCP was shown to be horn shaped and to specifically bind the upper domain of each major capsid protein in hexons but not in pentons. In Kaposi's sarcoma-associated herpesvirus (KSHV), the protein encoded by the ORF65 gene (pORF65) is the putative SCP but its location remains controversial due to the absence of such horn-shaped densities from both the pentons and hexons of the KSHV capsid reconstructions. To directly locate the KSHV SCP, we have used electron cryomicroscopy and three-dimensional reconstruction techniques to compare the three-dimensional structure of KSHV capsids to that of anti-pORF65 antibody-labeled capsids. Our difference map shows prominent antibody densities bound to the tips of the hexons but not to pentons, indicating that KSHV SCP is attached to the upper domain of the major capsid protein in hexons but not to that in pentons, similar to HSV-1 SCP. The lack of horn-shaped densities on the hexons indicates that KSHV SCP exhibits structural features that are substantially different from those of HSV-1 SCP. The location of SCP at the outermost regions of the capsid suggests a possible role in mediating capsid interactions with the tegument and cytoskeletal proteins during infection.     

Structure of the reovirus membrane-penetration protein, Mu1, in a complex with is protector protein, Sigma3

Cell entry by nonenveloped animal viruses requires membrane penetration without membrane fusion. The reovirus penetration agent is the outer-capsid protein, Mu1. The structure of Mu1, complexed with its "protector" protein, Sigma3, and the fit of this Mu1(3)Sigma3(3) heterohexameric complex into the cryoEM image of an intact virion, reveal molecular events essential for viral penetration. Autolytic cleavage divides Mu1 into myristoylated Mu1N and Mu1C. A long hydrophobic pocket can receive the myristoyl group. Dissociation of Mu1N, linked to a major conformational change of the entire Mu1 trimer, must precede myristoyl-group insertion into the cellular membrane. A myristoyl switch, coupling exposure of the fatty acid chain, autolytic cleavage of Mu1N, and long-range molecular rearrangement of Mu1C, thus appears to be part of the penetration mechanism.     

Different pathways for iNOS-mediated toxicity in vitro dependent on neuronal maturation and NMDA receptor expression

Co-localization of activated microglia and damaged neurones seen in brain injury suggests microglia-induced neurodegeneration. Activated microglia release two potential neurotoxins, excitatory amino acids and nitric oxide (NO), but their contribution to mechanisms of injury is poorly understood. Using co-cultures of rat microglia and embryonic cortical neurones, we show that inducible NO synthase (iNOS)-derived NO aloneis responsible for neuronal death from interferon gamma (IFNgamma) +lipopolysaccharide (LPS)-activated microglia. Neurones remain sensitive to NO irrespective of maturation state but, whereas blocking NMDA receptor activation with MK801 has no effect on NO-mediated toxicity to immature neurones, MK801 rescues 60-70% of neurones matured in culture for 12 days. Neuronal expression of NMDA receptors increases with maturation in culture, accounting for increased susceptibility to excitotoxins seen in more mature cultures. We show that MK801 delays the death of more mature neurones caused by the NO-donor DETA/NO indicating that NO elicits an excitotoxic mechanism, most likely through neuronal glutamate release. Thus, similar concentrations of nitric oxide cause neuronal death by two distinct mechanisms: NO acts directly upon immature neurones but indirectly, via NMDA receptors, on more mature neurones. Our results therefore extend existing evidence for NO-mediated toxicity and show a complex interaction between inflammatory and excitotoxic mechanisms of injury in mature neurones.     

FGF-dependent generation of oligodendrocytes by a hedgehog-independent pathway

During development, spinal cord oligodendrocyte precursors (OPCs) originate from the ventral, but not dorsal, neuroepithelium. Sonic hedgehog (SHH) has crucial effects on oligodendrocyte production in the ventral region of the spinal cord; however, less is known regarding SHH signalling and oligodendrocyte generation from neural stem cells (NSCs). We show that NSCs isolated from the dorsal spinal cord can generate oligodendrocytes following FGF2 treatment, a MAP kinase dependent phenomenon that is associated with induction of the obligate oligogenic gene Olig2. Cyclopamine, a potent inhibitor of hedgehog signalling, did not block the formation of oligodendrocytes from FGF2-treated neurosphere cultures. Furthermore, neurospheres generated from SHH null mice also produced oligodendrocytes, even in the presence of cyclopamine. These findings are compatible with the idea of a hedgehog independent pathway for oligodendrocyte generation from neural stem cells.     

Improved determination of urinary oxalate with alkylamine glass bound barley oxalate oxidase

The measurement of oxalate in urine was improved by employing barley oxalate oxidase immobilized on alkylamine glass beads affixed in a glass beaker. The minimum detection limit was 3.6 mg l(-1) urine. The recovery of added oxalate was 88.9+/-9.2%. Within- and between-assay coefficients of variation (CV) were <4.0 and <9.4%, respectively. The urinary oxalate values were obtained by a commercial kit method and the present method showed a good correlation (0.999). The method is free from tedious handling of glass beads and Cl- interference.     

Drug Repurposing: A Systematic Approach to Evaluate Candidate Oral Neuroprotective Interventions for Secondary Progressive Multiple Sclerosis

Objective

To develop and implement an evidence based framework to select, from drugs already licenced, candidate oral neuroprotective drugs to be tested in secondary progressive multiple sclerosis.

Design

Systematic review of clinical studies of oral putative neuroprotective therapies in MS and four other neurodegenerative diseases with shared pathological features, followed by systematic review and meta-analyses of the in vivo experimental data for those interventions. We presented summary data to an international multi-disciplinary committee, which assessed each drug in turn using pre-specified criteria including consideration of mechanism of action.

Results

We identified a short list of fifty-two candidate interventions. After review of all clinical and pre-clinical evidence we identified ibudilast, riluzole, amiloride, pirfenidone, fluoxetine, oxcarbazepine, and the polyunsaturated fatty-acid class (Linoleic Acid, Lipoic acid; Omega-3 fatty acid, Max EPA oil) as lead candidates for clinical evaluation.

Conclusions

We demonstrate a standardised and systematic approach to candidate identification for drug rescue and repurposing trials that can be applied widely to neurodegenerative disorders.     

MVA recombinants expressing the fusion and hemagglutinin genes of PPRV protects goats against virulent challenge

Peste des Petits Ruminants (PPR) is a highly contagious animal disease caused by the Peste des Petits Ruminants virus (PPRV) belonging to the genus morbillivirus and family Paramyxoviridae. The disease results in high morbidity and mortality in goats, sheep and in some small wild ruminants. The presence of large number of small ruminants reared in endemic areas makes PPR a notorious disease threatening the livelihood of poor farmers. Conventional vaccination using a live, attenuated vaccine gives adequate protection but cannot be used in case of eradication of the disease due to difficulty in differentiation of infected animals from the vaccinated ones.