Quantifying information transfer by protein domains: Analysis of the Fyn SH2 domain structure

Background  

Efficient communication between distant sites within a protein is essential for cooperative biological response. Although often associated with large allosteric movements, more subtle changes in protein dynamics can also induce long-range correlations. However, an appropriate formalism that directly relates protein structural dynamics to information exchange between functional sites is still lacking.     

Plasma progesterone concentrations during early pregnancy in spring- and autumn-bred ewes

The objective of this experiment was to measure blood progesterone concentrations during early gestation to determine if the apparent reproductive failure in ewes bred out-of-season is due to a failure to conceive or embryonic loss. Blood samples were collected from spring- (n=61) and autumn-bred ewes (n=29) from Days 8 to 39 post-oestrus. Serum progesterone concentrations were analysed to ascertain whether ewes were ovulating and failing to maintain pregnancy, or conception was failing. Following pregnancy diagnosis 62 days after ram introduction, ewes were categorised as; no display of oestrus, mated but then identified as non-pregnant, or pregnant. A majority of spring-bred ewes that failed to display oestrus had silent oestrus (86%) and 66% of those ewes had abnormally short-lived corpora lutea. Circulating progesterone concentrations during dioestrus in ewes that had ovulated and displayed oestrus were unaffected by season. Similarly, progesterone concentrations during dioestrus did not differ between pregnant and mated non-pregnant ewes. The results indicated that while early luteylosis, low progesterone secretion from corpora lutea and embryo mortality did occur, these were in only a small proportion of ewes. Progesterone concentrations indicated that a majority of mated non-pregnant ewes had elevated progesterone concentrations necessary for the production of at least one viable embryo/foetus. This may be indicative to the failure of maternal recognition of pregnancy, and it is recommended that events surrounding this stage of pregnancy (Days 12-14) be examined more closely in ewes during the non-breeding season.     

Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

ETISEQ – an algorithm for automated elution time ion sequencing of concurrently fragmented peptides for mass spectrometry-based proteomics

Background  

Concurrent peptide fragmentation (i.e. shotgun CID, parallel CID or MS^E) has emerged as an alternative to data-dependent acquisition in generating peptide fragmentation data in LC-MS/MS proteomics experiments. Concurrent peptide fragmentation data acquisition has been shown to be advantageous over data-dependent acquisition by providing greater detection dynamic range and providing more accurate quantitative information. Nevertheless, concurrent peptide fragmentation data acquisition remains to be widely adopted due to the lack of published algorithms designed specifically to process or interpret such data acquired on any mass spectrometer.     

Prostaglandin J2 alters pro-survival and pro-death gene expression patterns and 26 S proteasome assembly in human neuroblastoma cells

Many neurodegenerative disorders are characterized by two pathological hallmarks: progressive loss of neurons and occurrence of inclusion bodies containing ubiquitinated proteins. Inflammation may be critical to neurodegeneration associated with ubiquitin-protein aggregates. We previously showed that prostaglandin J2 (PGJ2), one of the endogenous products of inflammation, induces neuronal death and the accumulation of ubiquitinated proteins into distinct aggregates. We now report that temporal microarray analysis of human neuroblastoma SK-N-SH revealed that PGJ2 triggered a "repair" response including increased expression of heat shock, protein folding, stress response, detoxification and cysteine metabolism genes. PGJ2 also decreased expression of cell growth/maintenance genes and increased expression of apoptotic genes. Over time pro-death responses prevailed over pro-survival responses, leading to cellular demise. Furthermore, PGJ2 increased the expression of proteasome and other ubiquitin-proteasome pathway genes. This increase failed to overcome PGJ2 inhibition of 26 S proteasome activity. Ubiquitinated proteins are degraded by the 26 S proteasome, shown here to be the most active proteasomal form in SK-N-SH cells. We demonstrate that PGJ2 impairs 26 S proteasome assembly, which is an ATP-dependent process. PGJ2 perturbs mitochondrial function, which could be critical to the observed 26 S proteasome disassembly, suggesting a cross-talk between mitochondrial and proteasomal impairment. In conclusion neurotoxic products of inflammation, such as PGJ2, may play a role in neurodegenerative disorders associated with the aggregation of ubiquitinated proteins by impairing 26 S proteasome activity and inducing a chain of events that culminates in neuronal cell death. Temporal characterization of these events is relevant to understanding the underlying mechanisms and to identifying potential early biomarkers.     

Cultural isolation is greater than genetic isolation across an avian hybrid zone

Elucidating the relationship between genetic and cultural evolution is important in understanding speciation, as learned premating barriers might be involved in maintaining species differences. Here, we test this relationship by examining a widely recognized premating barrier, bird song, in a hybrid zone between black‐throated green (Setophaga virens) and Townsend's warblers (S. townsendi). We use song analysis, genomic techniques and playback experiments to characterize the cultural and genetic backgrounds of individuals in this region, expecting that if song is an important reproductive barrier between these species, there should be a strong relationship between song and genotype. We show that songs in the hybrid zone correspond to the distinctly different songs found in allopatry but that song and genotype are not tightly coupled in sympatry. Allopatric individuals responded only to local songs, indicating that individuals may have learned to respond to songs they commonly hear. We observed discordance between song and genotype clines; a narrower cline suggests that cultural selection on song is stronger than natural selection on genotype. These findings indicate that song is unlikely to play a role in reproductive isolation between these species, and we suggest that spatial variation in song may nonetheless be maintained by frequency‐dependent cultural selection. This decoupling of genes and culture may contribute to hybridization in this region.     

Extrapair paternity in golden-cheeked gibbons (Nomascus gabriellae) in the secondary lowland forest of Cat Tien National Park, Vietnam

We observed 18 groups of golden-cheeked gibbons (Nomascus gabriellae) in the secondary lowland forest in Cat Tien National Park from January 2004 to December 2005 to obtain information about the social organization of this little-studied species, in an area where the population is recovering through increased protection and forest regeneration. DNA from faecal samples of 10 infants and juveniles identified 1 case of extrapair paternity. DNA from faecal samples of 18 adults from three communities revealed the majority of adults sampled of both sexes to be related to adults in neighbouring territories. Overall, the indications are that in this empty habitat, gibbons appear to be able to establish territories adjacent to those of their kin.     

SHAPE analysis of the FIV Leader RNA reveals a structural switch potentially controlling viral packaging and genome dimerization

Feline immunodeficiency virus (FIV) infects many species of cat, and is related to HIV, causing a similar pathology. High-throughput selective 2' hydroxyl acylation analysed by primer extension (SHAPE), a technique that allows structural interrogation at each nucleotide, was used to map the secondary structure of the FIV packaging signal RNA. Previous studies of this RNA showed four conserved stem-loops, extensive long-range interactions (LRIs) and a small, palindromic stem-loop (SL5) within the gag open reading frame (ORF) that may act as a dimerization initiation site (DIS), enabling the virus to package two copies of its genome. Our analyses of wild-type (wt) and mutant RNAs suggest that although the four conserved stem-loops are static structures, the 5' and 3' regions previously shown to form LRI also adopt an alternative, yet similarly conserved conformation, in which the putative DIS is occluded, and which may thus favour translational and splicing functions over encapsidation. SHAPE and in vitro dimerization assays were used to examine SL5 mutants. Dimerization contacts appear to be made between palindromic loop sequences in SL5. As this stem-loop is located within the gag ORF, recognition of a dimeric RNA provides a possible mechanism for the specific packaging of genomic over spliced viral RNAs.