Adenosine monophosphate deaminase 3 activation shortens erythrocyte half-life and provides malaria resistance in mice.

The factors that determine red blood cell (RBC) lifespan and the rate of RBC aging have not been fully elucidated. In several genetic conditions, including sickle cell disease, thalassemia, and G6PD deficiency, erythrocyte lifespan is significantly shortened. Many of these diseases are also associated with protection from severe malaria, suggesting a role for accelerated RBC senescence and clearance in malaria resistance. Here, we report a novel, N-ethyl-N-nitrosourea-induced mutation that causes a gain of function in adenosine 5'-monophosphate deaminase (AMPD3). Mice carrying the mutation exhibit rapid RBC turnover, with increased erythropoiesis, dramatically shortened RBC lifespan, and signs of increased RBC senescence/eryptosis, suggesting a key role for AMPD3 in determining RBC half-life. Mice were also found to be resistant to infection with the rodent malaria Plasmodium chabaudi. We propose that resistance to P. chabaudi is mediated by increased RBC turnover and higher rates of erythropoiesis during infection.

Four species of arboreal folivore show differential tolerance to a secondary metabolite.

The marsupials that eat Eucalyptus in south-eastern Australia provide an example of animals with similar niche requirements occurring sympatrically. They certainly differ in size, ranging from about 1 kg in the greater glider (Petauroides volans) and the closely related common ringtail possum (Pseudocheirus peregrinus), to 4 kg (common brushtail possum, Trichosurus vulpecula) and up to 15 kg in the koala (Phascolarctos cinereus). All species, however, may eat considerable amounts of eucalypt foliage, often favouring the same species, and thus appear to compete for food. In order to better understand the degree of competition for food, we measured feeding by the greater glider in response to increasing concentrations of a specific group of eucalypt plant secondary metabolites (PSM), the sideroxylonals, and then compared it to results published for the other species. The greater glider was more resilient than the other species to increasing concentrations of sideroxylonals. We suggest this allows gliders to feed on leaves from the eucalypt subgenus, Symphyomyrtus, while its small size and gliding ability allow it to feed where koalas cannot, on the young leaves on top of the canopy. In contrast, the common ringtail possum is well adapted to feeding from species of the subgenus Eucalyptus, which do not produce sideroxylonals but contain less available nitrogen (AvailN) than do the symphyomyrtles. These 'nutritional niches' segregate the forest and along with other factors, such as generalist and specialist feeding strategies and differences in body size and requirements for shelter, presumably minimise competition between the marsupial species.

No evidence for genome editing in mouse zygotes and HEK293T human cell line using the DNA-guided Natronobacterium gregoryi Argonaute (NgAgo).

A recently published research article reported that the extreme halophile archaebacterium Natronobacterium gregoryi Argonaute enzyme (NgAgo) could cleave the cellular DNA under physiological temperature conditions in cell line and be implemented as an alternative to CRISPR/Cas9 genome editing technology. We assessed this claim in mouse zygotes for four loci (Sptb, Tet-1, Tet-2 and Tet-3) and in the human HEK293T cell line for the EMX1 locus. Over 100 zygotes were microinjected with nls-NgAgo-GK plasmid provided from Addgene and various concentrations of 5'-phosphorylated guide DNA (gDNA) from 2.5 ng/µl to 50 ng/µl and cultured to blastocyst stage of development. The presence of indels was verified using T7 endonuclease 1 assay (T7E1) and Sanger sequencing. We reported no evidence of successful editing of the mouse genome. We then assessed the lack of editing efficiency in HEK293T cell line for the EMX1 endogenous locus by monitoring the NgAgo protein expression level and the editing efficiency by T7E1 assay and Sanger sequencing. We reported that the NgAgo protein was expressed from 8 hours to a maximum expression at 48 hours post-transfection, confirming the efficient delivery of the plasmid and the gDNA but no evidence of successful editing of EMX1 target in all transfected samples. Together our findings indicate that we failed to edit using NgAgo.

GSTO1-1 plays a pro-inflammatory role in models of inflammation, colitis and obesity.

Glutathione transferase Omega 1 (GSTO1-1) is an atypical GST reported to play a pro-inflammatory role in response to LPS. Here we show that genetic knockout of Gsto1 alters the response of mice to three distinct inflammatory disease models. GSTO1-1 deficiency ameliorates the inflammatory response stimulated by LPS and attenuates the inflammatory impact of a high fat diet on glucose tolerance and insulin resistance. In contrast, GSTO1-1 deficient mice show a more severe inflammatory response and increased escape of bacteria from the colon into the lymphatic system in a dextran sodium sulfate mediated model of inflammatory bowel disease. These responses are similar to those of TLR4 and MyD88 deficient mice in these models and confirm that GSTO1-1 is critical for a TLR4-like pro-inflammatory response in vivo. In wild-type mice, we show that a small molecule inhibitor that covalently binds in the active site of GSTO1-1 can be used to ameliorate the inflammatory response to LPS. Our findings demonstrate the potential therapeutic utility of GSTO1-1 inhibitors in the modulation of inflammation and suggest their possible application in the treatment of a range of inflammatory conditions.

A protocol to express and isolate HRV16 3C protease for use in protease assays.

Human rhinovirus (HRV) proteases are highly conserved across serotypes and have very similar target specificity. However, there are some serotype-specific differences in their action. It is therefore necessary when performing in vitro protease assays to ensure that the recombinant proteases are specific to the serotype of the HRV under study. We describe a simple method for isolating HRV16 3C protease from a bacterial expression system, including transformation of bacterial cells with a commercially available cDNA plasmid which can be adapted to use for 3C proteases from any other HRV serotypes. The extracted, active 3C protease can then be used for in vitro protease assays.

Reverse genetic engineering of the human rhinovirus serotype 16 genome to introduce an antibody-detectable tag.

The ability to accurately detect viral proteins during infection is essential for virology research, and the lack of specific antibodies can make this detection difficult. Reverse genetic engineering of virus genomes to alter the wild-type genome is a powerful technique to introduce a detectable tag onto a viral protein. Here we outline a method to incorporate an influenza hemagglutinin epitope tag onto the 2A protease of HRV16. The method uses site-directed mutagenesis PCR to introduce the sequence for the HA antigen onto either the C or N termini of 2A protease while keeping the relevant internal cleavage sites intact. The new viral product is then cloned into a wild-type HRV16 plasmid and transfected into Ohio Hela cells to produce recombinant virus.

Variation in the nuclear effects of infection by different human rhinovirus serotypes.

Human rhinovirus (HRV) is a positive sense RNA virus, which, despite replicating in the cytoplasm, has a significant impact on nuclear transport and nuclear localization of host proteins. A number of studies have identified differences between HRV serotypes, with respect to host response, protease activity and replicative ability. Here we report the sero-specific effects of two group-A HRV serotypes, the minor group HRV2 and the major group HRV16, on nuclear transport and nuclear protein localization. Using Western analysis, immunofluorescence and real time PCR, we show that HRV2 replicates at a faster rate than HRV16, which correlates with earlier production of viral proteases and disruption of host nuclear transport. There is significant variation in the nuclear effects of different rhinovirus species, which in turn may impact disease progression and patient response.

Proteases of human rhinovirus: role in infection.

Human rhinoviruses (HRV) are the major etiological agents of the common cold and asthma exacerbations, with significant worldwide health and economic impact. Although large-scale population vaccination has proved successful in limiting or even eradicating many viruses, the more than 100 distinct serotypes mean that conventional vaccination is not a feasible strategy to combat HRV. An alternative strategy is to target conserved viral proteins such as the HRV proteases, 2A(pro) and 3C(pro), the focus of this review. Necessary for host cell shutoff, virus replication, and pathogenesis, 2A(pro) and 3C(pro) are clearly viable drug targets, and indeed, 3C(pro) has been successfully targeted for treating the common cold in experimental infection. 2A(pro) and 3C(pro) are crucial for virus replication due to their role in polyprotein processing as well as cleavage of key cellular proteins to inhibit cellular transcription and translation. Intriguingly, the action of the HRV proteases also disrupts nucleocytoplasmic trafficking, contributing to HRV cytopathic effects. Improved understanding of the protease-cell interactions should enable new therapeutic approaches to be identified for drug development.

The Relative Concentrations of Nutrients and Toxins Dictate Feeding by a Vertebrate Browser, the Greater Glider Petauroides volans.

Although ecologists believe that vertebrate herbivores must select a diet that allows them to meet their nutritional requirements, while avoiding intoxication by plant secondary metabolites, this is remarkably difficult to show. A long series of field and laboratory experiments means that we have a good understanding of the factors that affect feeding by leaf-eating marsupials. This knowledge and the natural intraspecific variation in Eucalyptus chemistry allowed us to test the hypothesis that the feeding decisions of greater gliders (Petauroides volans) depend on the concentrations of available nitrogen (incorporating total nitrogen, dry matter digestibility and tannins) and of formylated phloroglucinol compounds (FPCs), potent antifeedants unique to Eucalyptus. We offered captive greater gliders foliage from two species of Eucalyptus, E. viminalis and E. melliodora, which vary naturally in their concentrations of available nitrogen and FPCs. We then measured the amount of foliage eaten by each glider and compared this with our laboratory analyses of foliar total nitrogen, available nitrogen and FPCs for each tree offered. The concentration of FPCs was the main factor that determined how much gliders ate of E. viminalis and E. melliodora, but in gliders fed E. viminalis the concentration of available nitrogen was also a significant influence. In other words, greater gliders ate E. viminalis leaves with a particular combination of FPCs and available nitrogen that maximised the nutritional gain but minimised their ingestion of toxins. In contrast, the concentration of total nitrogen was not correlated with feeding. This study is among the first to empirically show that browsing herbivores select a diet that balances the potential gain (available nutrients) and the potential costs (plant secondary chemicals) of eating leaves. The major implication of the study is that it is essential to identify the limiting nutrients and relevant toxins in a system in order to understand feeding behaviour.