Small interfering RNAs against the TAR RNA binding protein, TRBP, a Dicer cofactor, inhibit human immunodeficiency virus type 1 long terminal repeat expression and viral production

RNA interference (RNAi) is now widely used for gene silencing in mammalian cells. The mechanism uses the RNA-induced silencing complex, in which Dicer, Ago2, and the human immunodeficiency virus type 1 (HIV-1) TAR RNA binding protein (TRBP) are the main components. TRBP is a protein that increases HIV-1 expression and replication by inhibition of the interferon-induced protein kinase PKR and by increasing translation of viral mRNA. After HIV infection, TRBP could restrict the viral RNA through its activity in RNAi or could contribute more to the enhancement of viral replication. To determine which function will be predominant in the virological context, we analyzed whether the inhibition of its expression could enhance or decrease HIV replication. We have generated small interfering RNAs (siRNAs) against TRBP and found that they decrease HIV-1 long terminal repeat (LTR) basal expression 2-fold, and the LTR Tat transactivated level up to 10-fold. In the context of HIV replication, siRNAs against TRBP decrease the expression of viral genes and inhibit viral production up to fivefold. The moderate increase in PKR expression and activation indicates that it contributes partially to viral gene inhibition. The moderate decrease in micro-RNA (miRNA) biogenesis by TRBP siRNAs suggests that in the context of HIV replication, TRBP functions other than RNAi are predominant. In addition, siRNAs against Dicer decrease viral production twofold and impede miRNA biogenesis. These results suggest that, in the context of HIV replication, TRBP contributes mainly to the enhancement of virus production and that Dicer does not mediate HIV restriction by RNAi.     

Genetic diversity within<Emphasis Type="Italic"> Pisum sativum</Emphasis> using protein- and PCR-based markers

A collection of 148 Pisum accessions, mostly from Western Europe, and including both primitive germplasm and cultivated types, was structured using 121 protein- and PCR-based markers. This molecular marker-based classification allowed us to trace back major lineages of pea breeding in Western Europe over the last decades, and to follow the main breeding objectives: increase of seed weight, introduction of the afila foliage type and white flowers, and improvement of frost tolerance for winter-sown peas. The classification was largely consistent with the available pedigree data, and clearly resolved the different main varietal types according to their end-uses (fodder, food and feed peas) from exotic types and wild forms. Fodder types were further separated into two sub-groups. Feed peas, corresponding to either spring-sown or winter-sown types, were also separated, with two apparently different gene pools for winter-sown peas. The garden pea group was the most difficult to structure, probably due to a continuum in breeding of feed peas from garden types. The classification also stressed the paradox between the narrowness of the genetic basis of recent cultivars and the very large diversity available within P. sativum. A sub-collection of 43 accessions representing 96% of the whole allelic variability is proposed as a starting point for the construction of a core collection.Communicated by C. Möllers     

Regeneration of plants from leaf explants of micropropagated clonal Eucalyptus grandis

Summary Callus production along with caulogenesis was obtained from leaf explants of micropropagated clonal Eucalyptus grandis after six to twelve weeks of culture. Out of eight clones tested, six were amenable to shoot production using simple media containing naphthaleneacetic acid and either 6-benzyladenine or zeatin. Differences in growth regulator requirements for organogenesis were observed between different clones. These shoots were then elongated on a medium containing gibberellic acid and rooted using media derived from the micropropagation medium. Light conditions were also found to be important for regeneration. This protocol is the first published on regeneration from nonseedling material and it will facilitate the Agrobacterium tumefaciens -mediated transformation of selected clonal Eucalyptus grandis.Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA naphthaleneacetic acid - PAR photosynthetically active radiation - PVP polyvinyl pyrrolidone - SH Schenk and Hildebrandt (1972) medium     

A genome-wide analysis of the flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.) dirigent protein family: from gene identification and evolution to differential regulation

Key message

Identification of DIR encoding genes in flax genome. Analysis of phylogeny, gene/protein structures and evolution. Identification of new conserved motifs linked to biochemical functions. Investigation of spatio-temporal gene expression and response to stress.

Abstract

Dirigent proteins (DIRs) were discovered during 8-8′ lignan biosynthesis studies, through identification of stereoselective coupling to afford either (+)- or (?)-pinoresinols from E-coniferyl alcohol. DIRs are also involved or potentially involved in terpenoid, allyl/propenyl phenol lignan, pterocarpan and lignin biosynthesis. DIRs have very large multigene families in different vascular plants including flax, with most still of unknown function. DIR studies typically focus on a small subset of genes and identification of biochemical/physiological functions. Herein, a genome-wide analysis and characterization of the predicted flax DIR 44-membered multigene family was performed, this species being a rich natural grain source of 8-8′ linked secoisolariciresinol-derived lignan oligomers. All predicted DIR sequences, including their promoters, were analyzed together with their public gene expression datasets. Expression patterns of selected DIRs were examined using qPCR, as well as through clustering analysis of DIR gene expression. These analyses further implicated roles for specific DIRs in (?)-pinoresinol formation in seed-coats, as well as (+)-pinoresinol in vegetative organs and/or specific responses to stress. Phylogeny and gene expression analysis segregated flax DIRs into six distinct clusters with new cluster-specific motifs identified. We propose that these findings can serve as a foundation to further systematically determine functions of DIRs, i.e. other than those already known in lignan biosynthesis in flax and other species. Given the differential expression profiles and inducibility of the flax DIR family, we provisionally propose that some DIR genes of unknown function could be involved in different aspects of secondary cell wall biosynthesis and plant defense.

     

Development of a new SSR-based linkage map in apricot and analysis of synteny with existing Prunus maps

Linkage maps of the apricot accessions ‘Lito’ and ‘BO 81604311’ were constructed using a total of 185 simple sequence repeat (SSR) markers sampled from those isolated in peach, almond, apricot and cherry; 74 were derived from a new apricot genomic library enriched for AG/CT microsatellite repeats (UDAp series), and in total, 98 had never been mapped in Prunus before. Eight linkage groups putatively corresponding to the eight haploid apricot chromosomes were identified for each parent. The two maps were 504 and 620 cM long, respectively, with 96 anchor markers showing a complete co-linearity between the two genomes. As few as three gaps larger than 15 cM were present in ‘Lito’ and six in the male parent; the maps align well with all the available SSR-based Prunus maps through the many common anchor loci. Only occasionally inverted positions between adjacent markers were found, and this can be explained by the small size of cross populations analysed in these Prunus maps and in those reported in literature. The newly developed apricot SSRs will help saturating the existing Prunus maps and will extend the choice of markers in the development of genetic maps for new breeding populations.     

Risk factors for asthma and allergy associated with urban migration: background and methodology of a cross-sectional study in Afro-Ecuadorian school children in Northeastern Ecuador (Esmeraldas-SCAALA Study)

Background

This study examined the attitudes and actions of 3415 physician-recruited adults aged ≥ 16 years with asthma in eleven countries who were prescribed regular maintenance therapy with inhaled corticosteroids or inhaled corticosteroids plus long-acting β₂-agonists.

Methods

Structured interviews were conducted to assess medication use, asthma control, and patients' ability to recognise and self-manage worsening asthma.

Results

Despite being prescribed regular maintenance therapy, 74% of patients used short-acting β₂-agonists daily and 51% were classified by the Asthma Control Questionnaire as having uncontrolled asthma. Even patients with well-controlled asthma reported an average of 6 worsenings/year. The mean period from the onset to the peak symptoms of a worsening was 5.1 days. Although most patients recognised the early signs of worsenings, the most common response was to increase short-acting β₂-agonist use; inhaled corticosteroids were increased to a lesser extent at the peak of a worsening.

Conclusion

Previous studies of this nature have also reported considerable patient morbidity, but in those studies approximately three-quarters of patients were not receiving regular maintenance therapy and not all had a physician-confirmed diagnosis of asthma. This study shows that patients with asthma receiving regular maintenance therapy still have high levels of inadequately controlled asthma. The study also shows that patients recognise deteriorating asthma control and adjust their medication during episodes of worsening. However, they often adjust treatment in an inappropriate manner, which represents a window of missed opportunity.     

Assessing species traits and landscape relationships of the mammalian carnivore community in a neotropical biological corridor

Mammalian carnivores play an important role in regulating food webs and ecosystems. While many carnivore populations are facing various threats such as habitat loss and fragmentation, poaching, and illegal trade, others have adapted to human-dominated landscapes. Information about Neotropical carnivore communities in particular is limited, especially in disturbed landscapes. We conducted a camera trap survey at 38 sites across the San Juan–La Selva Biological Corridor in Costa Rica to assess occupancy and detection probabilities of the carnivore community. We developed hypotheses within a likelihood-based framework in order to determine the landscape features and species traits (diet and size) that influenced their occupancy. We detected nine of the 13 native carnivores predicted to occur in the corridor. When modeled separately, each species responded to land cover changes differently, suggesting no strong community-wide predictors of occupancy. We then modeled three separate guilds within the carnivore community: omnivorous mesopredators, obligate carnivorous mesopredators, and apex predators. These community guild models revealed a negative relationship between omnivorous mesopredators and increasing forest and tree plantation cover, suggesting omnivores utilize forest fragments and edge habitats in agricultural landscapes. Obligate carnivorous mesopredator models did not reveal any strong habitat relationships, but landscape effects tended to contradict our a priori predictions. Apex predators were positively associated with increasing forest and tree plantation cover, protected areas, and increasing distances to villages. Alarmingly, apex predators and obligate carnivorous mesopredators were generally rare within the biological corridor. A lack of top-down control alone might result in heightened occupancy for all mesopredators, but because the community is dominated by omnivorous species, bottom-up release from human-induced land cover changes and resource provision may better explain their high occupancy.     

Respiratory allergy to Blomia tropicalis: Immune response in four syngeneic mouse strains and assessment of a low allergen-dose,short-term experimental model

Background

The dust mite Blomia tropicalis is an important source of aeroallergens in tropical areas. Although a mouse model for B. tropicalis extract (BtE)-induced asthma has been described, no study comparing different mouse strains in this asthma model has been reported. The relevance and reproducibility of experimental animal models of allergy depends on the genetic background of the animal, the molecular composition of the allergen and the experimental protocol.

Objectives

This work had two objectives. The first was to study the anti-B. tropicalis allergic responses in different mouse strains using a short-term model of respiratory allergy to BtE. This study included the comparison of the allergic responses elicited by BtE with those elicited by ovalbumin in mice of the strain that responded better to BtE sensitization. The second objective was to investigate whether the best responder mouse strain could be used in an experimental model of allergy employing relatively low BtE doses.

Methods

Groups of mice of four different syngeneic strains were sensitized subcutaneously with 100 μg of BtE on days 0 and 7 and challenged four times intranasally, at days 8, 10, 12, and 14, with 10 μg of BtE. A/J mice, that were the best responders to BtE sensitization, were used to compare the B. tropicalis-specific asthma experimental model with the conventional experimental model of ovalbumin (OVA)-specific asthma. A/J mice were also sensitized with a lower dose of BtE.

Results

Mice of all strains had lung inflammatory-cell infiltration and increased levels of anti-BtE IgE antibodies, but these responses were significantly more intense in A/J mice than in CBA/J, BALB/c or C57BL/6J mice. Immunization of A/J mice with BtE induced a more intense airway eosinophil influx, higher levels of total IgE, similar airway hyperreactivity to methacholine but less intense mucous production, and lower levels of specific IgE, IgG1 and IgG2 antibodies than sensitization with OVA. Finally, immunization with a relatively low BtE dose (10 μg per subcutaneous injection per mouse) was able to sensitize A/J mice, which were the best responders to high-dose BtE immunization, for the development of allergy-associated immune and lung inflammatory responses.

Conclusions

The described short-term model of BtE-induced allergic lung disease is reproducible in different syngeneic mouse strains, and mice of the A/J strain was the most responsive to it. In addition, it was shown that OVA and BtE induce quantitatively different immune responses in A/J mice and that the experimental model can be set up with low amounts of BtE.