Modification of picornavirus genomic RNA using ‘click’ chemistry shows that unlinking of the VPg peptide is dispensable for translation and replication of the incoming viral RNA

Picornaviruses constitute a large group of viruses comprising medically and economically important pathogens such as poliovirus, coxsackievirus, rhinovirus, enterovirus 71 and foot-and-mouth disease virus. A unique characteristic of these viruses is the use of a viral peptide (VPg) as primer for viral RNA synthesis. As a consequence, all newly formed viral RNA molecules possess a covalently linked VPg peptide. It is known that VPg is enzymatically released from the incoming viral RNA by a host protein, called TDP2, but it is still unclear whether the release of VPg is necessary to initiate RNA translation. To study the possible requirement of VPg release for RNA translation, we developed a novel method to modify the genomic viral RNA with VPg linked via a ‘non-cleavable’ bond. We coupled an azide-modified VPg peptide to an RNA primer harboring a cyclooctyne [bicyclo[6.1.0]nonyne (BCN)] by a copper-free ‘click’ reaction, leading to a VPg-triazole-RNA construct that was ‘non-cleavable’ by TDP2. We successfully ligated the VPg-RNA complex to the viral genomic RNA, directed by base pairing. We show that the lack of VPg unlinkase does not influence RNA translation or replication. Thus, the release of the VPg from the incoming viral RNA is not a prerequisite for RNA translation or replication.     

Winter habitat use does not influence spring arrival dates or the reproductive success of Yellow Warblers breeding in the arctic

Winter habitat use can influence the breeding success of migratory songbirds in temperate regions due to its impact on bird condition and breeding phenology. How such carry-over effects vary with latitude is unknown. To address this question, we examined how winter habitat use, inferred from δ¹³C and δ¹⁵N signatures in winter-grown feathers, influenced the breeding phenology and productivity of Yellow Warblers (Setophaga petechia) at the extreme north of their range in the Canadian arctic (68°N) and compared this population with midlatitude Yellow Warbler (51°N) and American Redstart (Setophaga ruticilla; 44°N) populations reported in previous studies. In the arctic, we examined male arrival dates, female clutch initiation dates and the relationship between these timing variables and the number and quality of offspring produced within the season. In contrast to warblers breeding at midlatitudes, we find no support for an impact of winter habitat use on breeding phenology or productivity. Male arrival dates and female clutch initiation dates in both young and older individuals were not correlated with isotopic signatures acquired on the wintering grounds. Males with enriched δ¹⁵N signatures paired more rapidly after arrival, indicating a possible relationship between winter habitat use and condition. This relationship did not enhance annual productivity for these individuals, however, as the negative relationship between breeding phenology and reproductive success in our arctic population was significantly weaker than among Yellow Warblers breeding further south. This reduction or absence of timing effects on productivity in the north effectively removes one pathway through which carry-over effects can act.     

StrainInfo introduces electronic passports for microorganisms

Microbiology builds upon biological material deposited in biological resource centers (BRCs) as a reference framework for collaborative research. BRCs assign so-called strain numbers to label the deposited material and are responsible for long-term preservation and worldwide distribution of the material. Cultured microorganisms can be deposited into multiple BRCs and BRCs also mutually exchange their holdings. As a result, many different strain numbers can be attached to biological material that stems from the same isolate. In practice, this material is considered equivalent and used interchangeably. This implies that finding information on given biological material requires all equivalent strain numbers to be used when searching.     

Life after treatment: detecting living microorganisms following exposure to UV light and chlorine dioxide

Rapid analytical methods are needed to quantify living microorganisms to determine if ships’ discharged ballast water is in compliance with national and international standards. Traditionally, regrowth assays and microscope counts of stained organisms—which are time-consuming, require expensive equipment, and require extensive staff training—are used to assess microorganisms. The goal of this study was to evaluate other approaches. Both ambient microorganisms from an oligotrophic marine environment and laboratory cultures of marine algae were evaluated following exposure to two types of ballast water treatment: ultraviolet (UV) light and chlorine dioxide (ClO₂). Microorganisms in two size classes (<10 and ≥10 to <50 μm) were quantified using regrowth assays and vital staining, and samples were evaluated using two rapid approaches: (1) chlorophyll a fluorescence and photochemical yield were measured using a pulse amplitude modulated fluorometer and (2) the concentration of adenosine triphosphate (ATP) was measured with a handheld luminometer. The response of microorganisms to UV and ClO₂ was evident in measurements of photochemical yield, as photochemical yield decreased at high doses. However, initial values of photochemical yield were variable and species-specific. Oddly, in some trials, initial fluorescence increased at intermediate UV doses; this phenomenon could lead to overestimation of total biomass. In samples treated with UV light, ATP was not significantly different among any of the doses used; however, concentrations of ATP were significantly lower at the highest dose of ClO₂ than control samples. These results demonstrate that approaches used for ballast water testing can be treatment-specific, and compliance approaches should be validated to determine their utility with the appropriate treatments.     

Re-Defining <Emphasis Type="Italic">Canis etruscus</Emphasis> (Canidae,Mammalia): A New Look into the Evolutionary History of Early Pleistocene Dogs Resulting from the Outstanding Fossil Record from Pantalla (Italy)

An outstanding sample of Canis etruscus has been found within the faunal assemblage from the early Pleistocene site of Pantalla (Italy), which is referred to the early late Villafranchian. Canis etruscus appeared in Europe about 2 Ma ago. It is regarded as an important taxon for biochronology, as its first occurrence (the “wolf event”) has been used to define one of the Villafranchian faunal turnovers. The discovery of four crania from Pantalla prompted a revision of C. etruscus, in order to better describe its cranial morphology. Since early studies, the distinction between C. etruscus and the coeval C. arnensis has been based mainly on mandibular traits. For this reason, our study is aimed at highlighting differences in craniodental characters between the two species. Canis arnensis has been conventionally considered a jackal-like dog, while C. etruscus is regarded as a wolf-like dog. Consequently, we decided to use jackals for comparison, in addition to C. lupus. Although the jackal group has been traditionally considered as quite homogenous (different species are partially sympatric and similar in both size and ecology), recent genetic studies demonstrate that jackals are not monophyletic. Considering the model offered by extant species, our goal is to delineate the degree of intra- and interspecific variability among the basal forms of the genus Canis.     

Rising sea level,temperature, and precipitation impact plant and ecosystem responses to elevated CO2 on a Chesapeake Bay wetland: review of a 28‐year study

An ongoing field study of the effects of elevated atmospheric CO₂ on a brackish wetland on Chesapeake Bay, started in 1987, is unique as the longest continually running investigation of the effects of elevated CO₂ on an ecosystem. Since the beginning of the study, atmospheric CO₂ increased 18%, sea level rose 20 cm, and growing season temperature varied with approximately the same range as predicted for global warming in the 21st century. This review looks back at this study for clues about how the effects of rising sea level, temperature, and precipitation interact with high atmospheric CO₂ to alter the physiology of C3 and C4 photosynthetic species, carbon assimilation, evapotranspiration, plant and ecosystem nitrogen, and distribution of plant communities in this brackish wetland. Rising sea level caused a shift to higher elevations in the Scirpus olneyi C3 populations on the wetland, displacing the Spartina patens C4 populations. Elevated CO₂ stimulated carbon assimilation in the Scirpus C3 species measured by increased shoot and root density and biomass, net ecosystem production, dissolved organic and inorganic carbon, and methane production. But elevated CO₂ also decreased biomass of the grass, S. patens C4. The elevated CO₂ treatment reduced tissue nitrogen concentration in shoots, roots, and total canopy nitrogen, which was associated with reduced ecosystem respiration. Net ecosystem production was mediated by precipitation through soil salinity: high salinity reduced the CO₂ effect on net ecosystem production, which was zero in years of severe drought. The elevated CO₂ stimulation of shoot density in the Scirpus C3 species was sustained throughout the 28 years of the study. Results from this study suggest that rising CO₂ can add substantial amounts of carbon to ecosystems through stimulation of carbon assimilation, increased root exudates to supply nitrogen fixation, reduced dark respiration, and improved water and nitrogen use efficiency.     

Allele-specific expression and eQTL analysis in mouse adipose tissue

Background

The simplest definition of cis-eQTLs versus trans, refers to genetic variants that affect expression in an allele specific manner, with implications on underlying mechanism. Yet, due to technical limitations of expression microarrays, the vast majority of eQTL studies performed in the last decade used a genomic distance based definition as a surrogate for cis, therefore exploring local rather than cis-eQTLs.

Results

In this study we use RNAseq to explore allele specific expression (ASE) in adipose tissue of male and female F1 mice, produced from reciprocal crosses of C57BL/6J and DBA/2J strains. Comparison of the identified cis-eQTLs, to local-eQTLs, that were obtained from adipose tissue expression in two previous population based studies in our laboratory, yields poor overlap between the two mapping approaches, while both local-eQTL studies show highly concordant results. Specifically, local-eQTL studies show ~60% overlap between themselves, while only 15-20% of local-eQTLs are identified as cis by ASE, and less than 50% of ASE genes are recovered in local-eQTL studies. Utilizing recently published ENCODE data, we also find that ASE genes show significant bias for SNPs prevalence in DNase I hypersensitive sites that is ASE direction specific.

Conclusions

We suggest a new approach to analysis of allele specific expression that is more sensitive and accurate than the commonly used fisher or chi-square statistics. Our analysis indicates that technical differences between the cis and local-eQTL approaches, such as differences in genomic background or sex specificity, account for relatively small fraction of the discrepancy. Therefore, we suggest that the differences between two eQTL mapping approaches may facilitate sorting of SNP-eQTL interactions into true cis and trans, and that a considerable portion of local-eQTL may actually represent trans interactions.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-471) contains supplementary material, which is available to authorized users.     

Bacillus subtilis spore protein SpoVAC functions as a mechanosensitive channel

A critical event during spore germination is the release of Ca‐DPA (calcium in complex with dipicolinic acid). The mechanism of release of Ca‐DPA through the inner membrane of the spore is not clear, but proteins encoded by the Bacillus subtilis spoVA operon are involved in the process. We cloned and expressed the spoVAC gene in Escherichia coli and characterized the SpoVAC protein. We show that SpoVAC protects E. coli against osmotic downshift, suggesting that it might act as a mechanosensitive channel. Purified SpoVAC was reconstituted in unilamellar lipid vesicles to determine the gating mechanism and pore properties of the protein. By means of a fluorescence‐dequenching assay, we show that SpoVAC is activated upon insertion into the membrane of the amphiphiles lysoPC and dodecylamine. Patch clamp experiments on E. coli giant spheroplast as well as giant unilamellar vesicles (GUVs) containing SpoVAC show that the protein forms transient pores with main conductance values of about 0.15 and 0.1 nS respectively. Overall, our data indicate that SpoVAC acts as a mechanosensitive channel and has properties that would allow the release of Ca‐DPA and amino acids during germination of the spore.     

Measuring Motivation for Appetitive Behaviour: Food-Restricted Broiler Breeder Chickens Cross a Water Barrier to Forage in an Area of Wood Shavings without Food

Broiler breeders (parents of meat chickens) are selected for fast growth and become obese if fed ad libitum. To avoid this and maintain good health and reproductive ability, they are feed restricted to about 1/3 of what they would eat ad libitum. As a result, they experience chronic hunger and exhibit abnormal behaviour patterns that may indicate stress and frustration. One approach to measuring hunger is to observe how much birds will work, such as pecking a key, for access to more or different types of food. However, the sight, smell, and feedback from consumption of the feed reward changes the context and may artificially raise feeding motivation. To avoid this, we tested broiler breeders in an apparatus in which they could work for access to a wooden platform covered in wood shavings by crossing a water runway which increased in length and depth in 8 successive tests. In the wood shavings area, they could perform exploratory and foraging behaviour (the appetitive phase of feeding) but were never rewarded with feed. Sixty birds were divided into three feed quantity treatments: commercial restriction (R), and twice (2R) or three times (3R) this amount. Overall, birds fed R worked harder to reach the wood shavings area (reached it in a larger number of tests) than 2R and 3R birds (P<0.001). More restricted birds took less time to reach the area (P<0.001, R<2R<3R) and spent more time foraging while there (P<0.001, R>2R>3R). This indicates that restricted-fed birds were hungry and willing to work for the opportunity to forage even though food was never provided, suggesting that their motivation to perform the appetitive component of feeding behaviour (foraging/food searching) was sufficient to sustain their response. Thus food restriction in broiler breeders is a welfare concern. However these methods could be used to test alternative feeding regimes to attempt to find ways of alleviating hunger while still maintaining healthy growth and reproduction in these birds.