Coronavirus NSP6 restricts autophagosome expansion

Autophagy is a cellular response to starvation that generates autophagosomes to carry long-lived proteins and cellular organelles to lysosomes for degradation. Activation of autophagy by viruses can provide an innate defense against infection, and for (+) strand RNA viruses autophagosomes can facilitate assembly of replicase proteins. We demonstrated that nonstructural protein (NSP) 6 of the avian coronavirus, infectious bronchitis virus (IBV), generates autophagosomes from the ER. A statistical analysis of MAP1LC3B puncta showed that NSP6 induced greater numbers of autophagosomes per cell compared with starvation, but the autophagosomes induced by NSP6 had smaller diameters compared with starvation controls. Small diameter autophagosomes were also induced by infection of cells with IBV, and by NSP6 proteins of MHV and SARS and NSP5, NSP6, and NSP7 of arterivirus PRRSV. Analysis of WIPI2 puncta induced by NSP6 suggests that NSP6 limits autophagosome diameter at the point of omegasome formation. IBV NSP6 also limited autophagosome and omegasome expansion in response to starvation and Torin1 and could therefore limit the size of autophagosomes induced following inhibition of MTOR signaling, as well as those induced independently by the NSP6 protein itself. MAP1LC3B-puncta induced by NSP6 contained SQSTM1, which suggests they can incorporate autophagy cargos. However, NSP6 inhibited the autophagosome/lysosome expansion normally seen following starvation. Taken together the results show that coronavirus NSP6 proteins limit autophagosome expansion, whether they are induced directly by the NSP6 protein, or indirectly by starvation or chemical inhibition of MTOR signaling. This may favor coronavirus infection by compromising the ability of autophagosomes to deliver viral components to lysosomes for degradation.     

Palliative Care among Heart Failure Patients in Primary Care: A Comparison to Cancer Patients Using English Family Practice Data

Introduction

Patients with heart failure have a significant symptom burden and other palliative care needs often over a longer period than patients with cancer. It is acknowledged that this need may be unmet but by how much has not been quantified in primary care data at the population level.

Methods

This was the first use of Clinical Practice Research Datalink, the world''s largest primary care database to explore recognition of the need for palliative care. Heart failure and cancer patients who had died in 2009 aged 18 or over and had at least one year of primary care records were identified. A palliative approach to care among patients with heart failure was compared to that among patients with cancer using entry onto a palliative care register as a marker for a palliative approach to care.

Results

Among patients with heart failure, 7% (234/3 122) were entered on the palliative care register compared to 48% (3 669/7 608) of cancer patients. Of heart failure patients on the palliative care register, 29% (69/234) were entered onto the register within a week of their death.

Conclusions

This confirms that the stark inequity in recognition of palliative care needs for people with heart failure in a large primary care dataset. We recommend a move away from prognosis based criteria for palliative care towards a patient centred approach, with assessment of and attention to palliative needs including advance care planning throughout the disease trajectory.     

Nutritional Correlates of Koala Persistence in a Low-Density Population

It is widely postulated that nutritional factors drive bottom-up, resource-based patterns in herbivore ecology and distribution. There is, however, much controversy over the roles of different plant constituents and how these influence individual herbivores and herbivore populations. The density of koala (Phascolarctos cinereus) populations varies widely and many attribute population trends to variation in the nutritional quality of the eucalypt leaves of their diet, but there is little evidence to support this hypothesis. We used a nested design that involved sampling of trees at two spatial scales to investigate how leaf chemistry influences free-living koalas from a low-density population in south east New South Wales, Australia. Using koala faecal pellets as a proxy for koala visitation to trees, we found an interaction between toxins and nutrients in leaves at a small spatial scale, whereby koalas preferred trees with leaves of higher concentrations of available nitrogen but lower concentrations of sideroxylonals (secondary metabolites found exclusively in eucalypts) compared to neighbouring trees of the same species. We argue that taxonomic and phenotypic diversity is likely to be important when foraging in habitats of low nutritional quality in providing diet choice to tradeoff nutrients and toxins and minimise movement costs. Our findings suggest that immediate nutritional concerns are an important priority of folivores in low-quality habitats and imply that nutritional limitations play an important role in constraining folivore populations. We show that, with a careful experimental design, it is possible to make inferences about populations of herbivores that exist at extremely low densities and thus achieve a better understanding about how plant composition influences herbivore ecology and persistence.     

SEX‐SPECIFIC PATTERNS OF MORPHOLOGICAL DIVERSIFICATION: EVOLUTION OF REACTION NORMS AND STATIC ALLOMETRIES IN NERIID FLIES

The consequences of sex‐specific selection for patterns of diversification remain poorly known. Because male secondary sexual traits are typically costly to express, and both costs and benefits are likely to depend on ambient environment and individual condition, such traits may be expected to diversify via changes in reaction norms as well as the scaling of trait size with body size (static allometry). We investigated morphological diversification within two species of Australian neriid flies (Telostylinus angusticollis, Telostylinus lineolatus) by rearing larvae from several populations on larval diets varying sixfold in nutrient concentration. Mean body size varied among populations of T. angusticollis, but body size reaction norms did not vary within either species. However, we detected diversification of reaction norms for body shape in males and females within both species. Moreover, unlike females, males also diversified in static allometry slope and reaction norms for static allometry slope of sexual and nonsexual traits. Our findings reveal qualitative sex differences in patterns of morphological diversification, whereby shape–size relationships diversify extensively in males, but remain conserved in females despite extensive evolution of trait means. Our results highlight the importance of incorporating plasticity and allometry in studies of adaptation and diversification.     

Conformational Changes in a Plant Ketol-Acid Reductoisomerase upon Mg and NADPH Binding as Revealed by Two Crystal Structures

Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) is an enzyme in the branched-chain amino acid biosynthesis pathway where it catalyzes the conversion of 2-acetolactate into (2R)-2,3-dihydroxy-3-isovalerate or the conversion of 2-aceto-2-hydroxybutyrate into (2R,3R)-2,3-dihydroxy-3-methylvalerate. KARI catalyzes two reactions—alkyl migration and reduction—and requires Mg²⁺ and NADPH for activity. To date, the only reported structures for a plant KARI are those of the spinach enzyme-Mn²⁺-(phospho)ADP ribose-(2R,3R)-2,3-dihydroxy-3-methylvalerate complex and the spinach KARI-Mg²⁺-NADPH-N-hydroxy-N-isopropyloxamate complex, where N-hydroxy-N-isopropyloxamate is a predicted transition-state analog. These studies demonstrated that the enzyme consists of two domains, N-domain and C-domain, with the active site at the interface of these domains. Here, we have determined the structures of the rice KARI-Mg²⁺ and rice KARI-Mg²⁺-NADPH complexes to 1.55 Å and 2.80 Å resolutions, respectively. In comparing the structures of all the complexes, several differences are observed. Firstly, the N-domain is rotated up to 15° relative to the C-domain, expanding the active site by up to 4 Å. Secondly, an α-helix in the C-domain that includes residues V510-T519 and forms part of the active site moves by ∼ 3.9 Å upon binding of NADPH. Thirdly, the 15 C-terminal amino acid residues in the rice KARI-Mg²⁺ complex are disordered. In the rice KARI-Mg²⁺-NADPH complex and the spinach KARI structures, many of the 15 residues bind to NADPH and the N-domain and cover the active site. Fourthly, the location of the metal ions within the active site can vary by up to 2.7 Å. The new structures allow us to propose that an induced-fit mechanism operates to (i) allow substrate to enter the active site, (ii) close over the active site during catalysis, and (iii) open the active site to facilitate product release.     

Direct Binding of Glyceraldehyde 3-Phosphate Dehydrogenase to Telomeric DNA Protects Telomeres against Chemotherapy-Induced Rapid Degradation

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that displays several non-glycolytic activities, including the maintenance and/or protection of telomeres. In this study, we determined the molecular mechanism and biological role of the interaction between GAPDH and human telomeric DNA. Using gel-shift assays, we show that recombinant GAPDH binds directly with high affinity (K_d = 45 nM) to a single-stranded oligonucleotide comprising three telomeric DNA repeats, and that nucleotides T1, G5, and G6 of the TTAGGG repeat are essential for binding. The stoichiometry of the interaction is 2:1 (DNA:GAPDH), and GAPDH appears to form a high-molecular-weight complex when bound to the oligonucleotide. Mutation of Asp32 and Cys149, which are localized to the NAD-binding site and the active-site center of GAPDH, respectively, produced mutants that almost completely lost their telomere-binding functions both in vitro and in situ (in A549 human lung cancer cells). Treatment of A549 cells with the chemotherapeutic agents gemcitabine and doxorubicin resulted in increased nuclear localization of expressed wild-type GAPDH, where it protected telomeres against rapid degradation, concomitant with increased resistance to the growth-inhibitory effects of these drugs. The non-DNA-binding mutants of GAPDH also localized to the nucleus when expressed in A549 cells, but did not confer any significant protection of telomeres against chemotherapy-induced degradation or growth inhibition; this occurred without the involvement of caspase activation or apoptosis regulation. Overall, these data demonstrate that GAPDH binds telomeric DNA directly in vitro and may have a biological role in the protection of telomeres against rapid degradation in response to chemotherapeutic agents in A549 human lung cancer cells.     

Response of birds to climatic variability; evidence from the western fringe of Europe

Ireland’s geographic location on the western fringe of the European continent, together with its island status and impoverished avifauna, provides a unique opportunity to observe changes in bird migration and distribution patterns in response to changing climatic conditions. Spring temperatures have increased in western Europe over the past 30 years in line with reported global warming. These have been shown, at least in part, to be responsible for changes in the timing of life cycle events (phenology) of plants and animals. In order to investigate the response of bird species in Ireland to changes in temperature, we examined ornithological records of trans-Saharan migrants over the 31-year period 1969–1999. Analysis of the data revealed that two discrete climatic phenomena produced different responses in summer migrant bird species. Firstly, a number of long-distance migrants showed a significant trend towards earlier arrival. This trend was evident in some species and was found to be a response to increasing spring air temperature particularly in the month of March. Secondly, (1) a step change in the pattern of occurrences of non-breeding migrant bird species, and (2) an increase in the ringing data of migrant species were found to correlate with a step change in temperature in 1987–1988. These results indicate that, for migrant bird species, the impact of a sudden change in temperature can be as important as any long-term monotonic trend, and we suggest that the impact of step change events merits further investigation on a wider range of species and across a greater geographical range.     

Increased expression of interferon-inducible genes in macaque lung tissues during simian immunodeficiency virus infection

Pulmonary infections and dysfunction are frequent outcomes during the development of immunodeficiency associated with human immunodeficiency virus type 1 (HIV-1) infection, and obtaining a better understanding of the immunologic changes that occur in lungs following HIV-1 infection will provide a foundation for the development of further intervention strategies. We sought here to identify changes in the pulmonary immune environment that arise during simian immunodeficiency virus (SIV) infection of rhesus macaques, which serves as an excellent model system for HIV-1 infection and disease. To examine the gene expression profiles of macaque lung tissues following infection with the pathogenic SIV/DeltaB670 isolate, we performed cDNA microarray hybridizations with lung total RNAs using two commercially available cDNA arrays and a custom-fabricated, immunologically focused macaque cDNA microarray. In situ hybridization and real-time RT-PCR were performed to provide additional analyses of gene expression. Among the genes exhibiting the highest level of induction in lung tissues were the IFN-gamma-inducible chemokines, CXCL10/IP-10 and CXCL9/Mig. In situ hybridization and real-time RT-PCR strongly supported these findings. Correlation analyses revealed that the levels of expression of IFN-gamma, CXCL9/Mig, and CXCL10/IP-10 mRNAs were all strongly positively correlated, and that CXCL10/IP-10 mRNA and Pneumocystis carinii rRNA were positively correlated. Taken together, these findings demonstrate that inflammatory chemokines are among the most differentially expressed mRNAs in macaque lung tissues during systemic SIV infection of rhesus macaques, and provide insight into the complicated events occurring in the lung tissues during HIV-1 infection in humans.