Commonly used caspase inhibitors designed based on substrate specificity profiles lack selectivity

Caspase regulation and activation have been extensively studied since the discovery of this class of proteases almost two decades ago, yet surprisingly few tools are available that can be used to monitor individual caspase activities [ 1 ]. The most commonly used tools include caspase-specific anti-sera as well as fluorogenic substrates and inhibitors. Unfortunately, antibody reagents often do not provide an accurate measure of caspase activity since several caspase family members （caspases 8/10 and 9） do not require proteolytic processing for activation [2, 3]. Furthermore, recent evidence suggests that caspase-7 （an executioner caspase） activation occurs via a catalytically active full-length intermediate that cannot be differentiated from the non-cleaved inactive zymogen using antibodies [4, 5].     

Activation of vascular endothelial nitric oxide synthase and heme oxygenase-1 expression by electrophilic nitro-fatty acids

Reactive oxygen species mediate a decrease in nitric oxide (NO) bioavailability and endothelial dysfunction, with secondary oxidized and nitrated by-products of these reactions contributing to the pathogenesis of numerous vascular diseases. While oxidized lipids and lipoproteins exacerbate inflammatory reactions in the vasculature, in stark contrast the nitration of polyunsaturated fatty acids and complex lipids yields electrophilic products that exhibit pluripotent anti-inflammatory signaling capabilities acting via both cGMP-dependent and -independent mechanisms. Herein we report that nitro-oleic acid (OA-NO₂) treatment increases expression of endothelial nitric oxide synthase (eNOS) and heme oxygenase 1 (HO-1) in the vasculature, thus transducing vascular protective effects associated with enhanced NO production. Administration of OA-NO₂ via osmotic pump results in a significant increase in eNOS and HO-1 mRNA in mouse aortas. Moreover, HPLC-MS/MS analysis showed that NO₂-FAs are rapidly metabolized in cultured endothelial cells (ECs) and treatment with NO₂-FAs stimulated the phosphorylation of eNOS at Ser¹¹⁷⁹. These posttranslational modifications of eNOS, in concert with elevated eNOS gene expression, contributed to an increase in endothelial NO production. In aggregate, OA-NO₂-induced eNOS and HO-1 expression by vascular cells can induce beneficial effects on endothelial function and provide a new strategy for treating various vascular inflammatory and hypertensive disorders.     

Old and new inhibitors of quinone reductase 2

Quinone reductase 2 is a cytosolic enzyme which catalyses the reduction of quinones, such as menadione and coenzymes Q. Despite a relatively close sequence-based resemblance to NAD(P)H:quinone oxidoreductase 1 (QR1), it has many different features. QR2 is the third melatonin binding site (MT3). It is inhibited in the micromolar range by melatonin, and does not accept conventional phosphorylated nicotinamides as hydride donors. QR2 has a powerful capacity to activate quinones leading to unexpected toxicity situations. In the present paper, we report the characterization of three QR2 modulators: melatonin, resveratrol and S29434. The latter compound inhibits QR2 activity with an IC₅₀ in the low nanomolar range. The potency of the modulators ranged as follows, from the least to the most potent: melatonin < resveratrol < S29434. These molecular tools might permit to explore and better understand the relationship existing between QR2 catalytic activity and the various pathological situations in which QR2 has a key role.     

A trans-Complementing Recombination Trap Demonstrates a Low Propensity of Flaviviruses for Intermolecular Recombination

Intermolecular recombination between the genomes of closely related RNA viruses can result in the emergence of novel strains with altered pathogenic potential and antigenicity. Although recombination between flavivirus genomes has never been demonstrated experimentally, the potential risk of generating undesirable recombinants has nevertheless been a matter of concern and controversy with respect to the development of live flavivirus vaccines. As an experimental system for investigating the ability of flavivirus genomes to recombine, we developed a “recombination trap,” which was designed to allow the products of rare recombination events to be selected and amplified. To do this, we established reciprocal packaging systems consisting of pairs of self-replicating subgenomic RNAs (replicons) derived from tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) that could complement each other in trans and thus be propagated together in cell culture over multiple passages. Any infectious viruses with intact, full-length genomes that were generated by recombination of the two replicons would be selected and enriched by end point dilution passage, as was demonstrated in a spiking experiment in which a small amount of wild-type virus was mixed with the packaged replicons. Using the recombination trap and the JEV system, we detected two aberrant recombination events, both of which yielded unnatural genomes containing duplications. Infectious clones of both of these genomes yielded viruses with impaired growth properties. Despite the fact that the replicon pairs shared approximately 600 nucleotides of identical sequence where a precise homologous crossover event would have yielded a wild-type genome, this was not observed in any of these systems, and the TBEV and WNV systems did not yield any viable recombinant genomes at all. Our results show that intergenomic recombination can occur in the structural region of flaviviruses but that its frequency appears to be very low and that therefore it probably does not represent a major risk in the use of live, attenuated flavivirus vaccines.RNA viruses are able to undergo rapid genetic changes in order to adapt to new hosts or environments. Although much of this flexibility is due to the error-prone nature of the RNA-dependent RNA polymerase, which generates an array of different point mutations within the viral population (23), recombination is also a common and important mechanism for generating viral diversity (18, 31, 42, 58). Recombination occurs when the RNA-dependent RNA polymerase switches templates during replication, an event that is favored when both templates share identical or very similar sequences. Three types of RNA recombination have been identified: homologous recombination occurs at sites with exact sequence matches; aberrant homologous recombination requires sequence homology, but crossover occurs either upstream or downstream of the site of homology, resulting in a duplication or deletion; and nonhomologous (or illegitimate) recombination is independent of sequence homology (31, 42).When the same cell is infected by viruses of two different strains, or even different species, recombination between their genomic RNAs can potentially lead to the emergence of new pathogens. A case in point is the emergence of Western equine encephalitis virus, a member of the genus Alphavirus, family Togaviridae, which arose by homologous recombination between Eastern equine encephalitis virus and Sindbis virus (14).Some mammalian RNA viruses can recombine at a frequency that is detectable in experimental settings (1, 2, 55), and phylogenetic analysis of partial or complete genome sequences suggests that RNA recombination is a widespread phenomenon. Naturally occurring recombinant viruses have been identified in almost every family of positive-stranded RNA viruses (31, 58).Flaviviruses are members of the genus Flavivirus, family Flaviviridae, a family that also includes the genera Pestivirus and Hepacivirus. Several of the flaviviruses are important human pathogens, such as Japanese encephalitis virus (JEV), West Nile virus (WNV), the dengue viruses, yellow fever virus, and tick-borne encephalitis virus (TBEV).Although there has never been a report of a pathogenic flavivirus strain arising due to recombination involving attenuated vaccine strains (39), the urgent necessity to develop tetravalent vaccines containing all four serotypes of dengue virus—two such vaccines are currently undergoing clinical testing (45)—has recently brought the recombination issue to the forefront of discussion among researchers, regulators, and vaccine producers (39). It has been suggested that recombination, either between the strains present in a multivalent vaccine or between an attenuated vaccine strain and a wild-type strain, could lead to the emergence of new viruses with unpredictable properties (49).So far, recombination between flavivirus genomes has not been demonstrated directly in the laboratory. However, phylogenetic analysis of partial genome sequences available in the GenBank database has suggested that homologous recombination may have occurred between closely related strains of dengue virus (20, 52, 54, 59). An experimental approach for assessing the ability of flavivirus genomes to recombine is therefore urgently needed.Flavivirus virions are composed of a single-stranded, positive-sense RNA genome that, together with the capsid protein C, forms the viral nucleocapsid. The nucleocapsid is covered by a lipid envelope containing the surface glycoproteins prM and E. These glycoproteins drive budding at the membrane of the endoplasmic reticulum during the assembly stage and mediate entry of the virus into host cells (41). Replicons, defined as self-replicating, noninfectious RNA molecules, can be generated by deleting parts or all of the region coding for the structural proteins C, prM, and E from the viral genome but maintaining all seven of the nonstructural proteins and the flanking noncoding sequences, which are required in cis for RNA replication (25). By providing the missing structural protein components in trans, replicons can be packaged into virus-like particles that are capable of a single round of infection (10, 15, 24, 47).Typically, researchers developing novel replicating vaccines, especially ones that involve multiple components, make an effort to come up with strategies to prevent recombination, for example by “wobbling” codons, i.e., replacing codons in homologous regions with synonymous ones encoding the same amino acid but consisting of a different nucleotide triplet (50, 57). In this study, in order to assess the propensity of flavivirus genomes to recombine, we took an opposite approach, establishing a “recombination trap” that favors the selection and sensitive detection of recombination products. This system takes advantage of the ability of replicon pairs containing deletions in their structural protein genes to complement each other in trans and thus be propagated together in cell culture, and by passage at limiting dilutions, it allows infectious RNA genomes arising by recombination between the two replicons to be preferentially selected.Using the recombination trap, we have now obtained the first direct evidence of recombination between flavivirus genomes in the laboratory. Aberrant homologous recombination was observed twice with JEV replicons, resulting in viruses with unnatural gene arrangements and reduced growth properties compared to those of wild-type JEV. No infectious recombinants of any kind were obtained when TBEV or WNV replicons were used. Interestingly, we never detected a fully infectious wild-type genome arising by homologous recombination in any of these systems. The results of this study show that the propensity of flavivirus genomes to recombine in the region coding for the structural proteins appears to be quite low, suggesting that recombination does not represent a major risk in the use of live, attenuated flavivirus vaccines.     

Experimental infection of self-cured Leiopelma archeyi with the amphibian chytrid Batrachochytrium dendrobatidis

The susceptibility of Archey's frog Leiopelma archeyi to Batrachochytrium dendrobatidis (Bd) is unknown, although one large population is thought to have declined sharply due to chytridiomycosis. As primary infection experiments were not permitted in this endangered New Zealand species, 6 wild-caught L. archeyi that naturally cleared infections with Bd while in captivity were exposed again to Bd to assess their immunity. These frogs were from an infected population at Whareorino, which has no known declines. All 6 L. archeyi became reinfected at low intensities, but rapidly self cured, most by 2 wk. Six Litoria ewingii were used as positive controls and developed heavier infections and clinical signs by 3 wk, demonstrating that the zoospore inoculum was virulent. Six negative controls of each species remained uninfected and healthy. Our results show that L. archeyi that have self cured have resistance to chytridiomycosis when exposed. The pattern is consistent with innate or acquired immunity to Bd, and immunological studies are needed to confirm this.     

Application of the survey protocol for chytridiomycosis to Queensland, Australia

Spread of the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), which causes chytridiomycosis, has resulted in the extinction of frogs, but the distribution of Bd is incompletely known. We trialled the survey protocol for Bd by attempting to systematically map its distribution in Queensland, Australia. Bd was easily detected in known infected areas, such as the Wet Tropics and South East Queensland. It was not detected in bioregions adjacent to, but inland from or to the north of, infected regions: Einasleigh Uplands and Cape York adjacent to the infected Wet Tropics; and Brigalow Belt South adjacent to the infected South East Queensland bioregion. These regions where Bd was not detected have bordered infected regions for between 15 yr (in northern Queensland) and 30 yr (in southern Queensland), and so they define the geographical limits of Bd with regard to the long-term environmental conditions in Queensland. The Gulf Plains, a bioregion distant from infected bioregions, was also negative. Bd was confined to rainforest and bordering habitats, such as wet eucalypt forests. Infections were largely confined to permanent water-associated species, consistent with this being an important cause of this group having the greatest declines. Our data supports biogeographic climatic models that show much of inland and northern Australia to be too hot and dry to support Bd. As there is limited opportunity for Bd to spread further in Queensland, the priority for management is reducing the impact of Bd in affected populations and assisting frogs to disperse into their former distributions. Given that the survey protocol has been applied successfully in Australia it may be useful for mapping the distribution of Bd in other parts of the world.