Biochemical Requirements for Two Dicer-Like Activities from Wheat Germ

RNA silencing pathways were first discovered in plants. Through genetic analysis, it has been established that the key silencing components called Dicer-like (DCL) genes have been shown to cooperatively process RNA substrates of multiple origin into distinct 21, 22 and 24 nt small RNAs. However, only few detailed biochemical analysis of the corresponding complexes has been carried out in plants, mainly due to the large unstable complexes that are hard to obtain or reconstitute in heterologous systems. Reconstitution of activity needs thorough understanding of all protein partners in the complex, something that is still an ongoing process in plant systems. Here, we use biochemical analysis to uncover properties of two previously identified native dicer-like activities from wheat germ. We find that standard wheat germ extract contains Dicer-like enzymes that convert double-stranded RNA (dsRNA) into two classes of small interfering RNAs of 21 and 24 nt in size. The 21 nt dicing activity, likely an siRNA producing complex known as DCL4, is 950 kDa-1.2 mDa in size and is highly unstable during purification processes but has a rather vast range for activity. On the contrary, the 24 nt dicing complex, likely the DCL3 activity, is relatively stable and comparatively smaller in size, but has stricter conditions for effective processing of dsRNA substrates. While both activities could process completely complementary dsRNA albeit with varying abilities, we show that DCL3-like 24 nt producing activity is equally good in processing incompletely complementary RNAs.     

Plasticity of amyloid fibrils

In experiments designed to characterize the basis of amyloid fibril stability through mutational analysis of the Abeta (1-40) molecule, fibrils exhibit consistent, significant structural malleability. In these results, and in other properties, amyloid fibrils appear to more resemble plastic materials generated from synthetic polymers than globular proteins. Thus, like synthetic polymers and plastics, amyloid fibrils exhibit both polymorphism, the ability of one polypeptide to form aggregates of different morphologies, and isomorphism, the ability of different polypeptides to grow into a fibrillar amyloid morphology. This view links amyloid with the prehistorical and 20th century use of proteins as starting materials to make films, fibers, and plastics, and with the classic protein fiber stretching experiments of the Astbury group. Viewing amyloids from the point of view of the polymer chemist may shed new light on a number of issues, such as the role of protofibrils in the mechanism of amyloid formation, the biological potency of fibrils, and the prospects for discovering inhibitors of amyloid fibril formation.     

Diversity,expression and mRNA targeting abilities of Argonaute-targeting miRNAs among selected vascular plants

Background

Micro (mi)RNAs are important regulators of plant development. Across plant lineages, Dicer-like 1 (DCL1) proteins process long ds-like structures to produce micro (mi) RNA duplexes in a stepwise manner. These miRNAs are incorporated into Argonaute (AGO) proteins and influence expression of RNAs that have sequence complementarity with miRNAs. Expression levels of AGOs are greatly regulated by plants in order to minimize unwarranted perturbations using miRNAs to target mRNAs coding for AGOs. AGOs may also have high promoter specificity-sometimes expression of AGO can be limited to just a few cells in a plant. Viral pathogens utilize various means to counter antiviral roles of AGOs including hijacking the host encoded miRNAs to target AGOs. Two host encoded miRNAs namely miR168 and miR403 that target AGOs have been described in the model plant Arabidopsis and such a mechanism is thought to be well conserved across plants because AGO sequences are well conserved.

Results

We show that the interaction between AGO mRNAs and miRNAs is species-specific due to the diversity in sequences of two miRNAs that target AGOs, sequence diversity among corresponding target regions in AGO mRNAs and variable expression levels of these miRNAs among vascular plants. We used miRNA sequences from 68 plant species representing 31 plant families for this analysis. Sequences of miR168 and miR403 are not conserved among plant lineages, but surprisingly they differ drastically in their sequence diversity and expression levels even among closely related plants. Variation in miR168 expression among plants correlates well with secondary structures/length of loop sequences of their precursors.

Conclusions

Our data indicates a complex AGO targeting interaction among plant lineages due to miRNA sequence diversity and sequences of miRNA targeting regions among AGO mRNAs, thus leading to the assumption that the perturbations by viruses that use host miRNAs to target antiviral AGOs can only be species-specific. We also show that rapid evolution and likely loss of expression of miR168 isoforms in tobacco is related to the insertion of MITE-like transposons between miRNA and miRNA* sequences, a possible mechanism showing how miRNAs are lost in few plant lineages even though other close relatives have abundantly expressing miRNAs.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1049) contains supplementary material, which is available to authorized users.     

Prevalence of Seropositivity to Pandemic Influenza A/H1N1 Virus in the United States following the 2009 Pandemic

Background

2009 pandemic influenza A/H1N1 (A(H1N1)pdm09) was first detected in the United States in April 2009 and resulted in a global pandemic. We conducted a serologic survey to estimate the cumulative incidence of A(H1N1)pdm09 through the end of 2009 when pandemic activity had waned in the United States.

Methods

We conducted a pair of cross sectional serologic surveys before and after the spring/fall waves of the pandemic for evidence of seropositivity (titer ≥40) using the hemagglutination inhibition (HI) assay. We tested a baseline sample of 1,142 serum specimens from the 2007–2008 National Health and Nutrition Examination Survey (NHANES), and 2,759 serum specimens submitted for routine screening to clinical diagnostic laboratories from ten representative sites.

Results

The age-adjusted prevalence of seropositivity to A(H1N1)pdm09 by year-end 2009 was 36.9% (95%CI: 31.7–42.2%). After adjusting for baseline cross-reactive antibody, pandemic vaccination coverage and the sensitivity/specificity of the HI assay, we estimate that 20.2% (95%CI: 10.1–28.3%) of the population was infected with A(H1N1)pdm09 by December 2009, including 53.3% (95%CI: 39.0–67.1%) of children aged 5–17 years.

Conclusions

By December 2009, approximately one-fifth of the US population, or 61.9 million persons, may have been infected with A(H1N1)pdm09, including around half of school-aged children.     

Facultative apomixis and hybridization in arctic Potentilla section Niveae (Rosaceae) from Svalbard

The degree of sexual seed set and the ability to cross were investigated in three taxa of Potentilla section Niveae ( P. chamissonis , P. insularis and P. nivea ) from the Svalbard Archipelago. Emasculated and bagged flowers had little or no seed set, while 71% of the emasculated, bagged and pollinated flowers had some. The taxa are interpreted as pseudogamous apomicts. Parental plants and offspring were subjected to isozyme analysis. Most of the offspring were clones of the mother plant, but 1.7% had bands from the pollen donor. The sexual offspring were all produced by mother plants of P. insularis , which is interpreted as a facultative apomict. Surprisingly, crosses between different taxa gave higher seed to ovule ratios and numbers of sexually produced offspring than crosses within taxa. Some of the sexually produced hybrid offspring had the same kind of hairs on the petioles as their mother taxon, showing that hybrids may not be intermediate in hair characters, which are considered important in the delimitation of these taxa. The results of the present study indicate very close relationships among the investigated plants.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 142 , 373−381.     

Methylglyoxal detoxification by a DJ-1 family protein provides dual abiotic and biotic stress tolerance in transgenic plants

Methylglyoxal (MG) is a key signaling molecule resulting from glycolysis and other metabolic pathways. During abiotic stress, MG levels accumulate to toxic levels in affected cells. However, MG is routinely detoxified through the action of DJ1/PARK7/Hsp31 proteins that are highly conserved across kingdoms and mutations in such genes are associated with neurodegenerative diseases. Here, we report for the first time that, similar to abiotic stresses, MG levels increase during biotic stresses in plants, likely contributing to enhanced susceptibility to a wide range of stresses. We show that overexpression of yeast Heat shock protein 31 (Hsp31), a DJ-1 homolog with robust MG detoxifying capabilities, confers dual biotic and abiotic stress tolerance in model plant Nicotiana tabacum. Strikingly, overexpression of Hsp31 in tobacco imparts robust stress tolerance against diverse biotic stress inducers such as viruses, bacteria and fungi, in addition to tolerance against a range of abiotic stress inducers. During stress, Hsp31 was targeted to mitochondria and induced expression of key stress-related genes. These results indicate that Hsp31 is a novel attractive tool to engineer plants against both biotic and abiotic stresses.     

An intersheet packing interaction in A beta fibrils mapped by disulfide cross-linking

Most models for the central cross-beta folding unit in amyloid fibrils of the Alzheimer's plaque protein Abeta align the peptides in register in H-bonded, parallel beta-sheet structure. Some models require the Abeta peptide to undergo a chain reversal when folding into the amyloid core, while other models feature very long extended chains, or zigzag chains, traversing the protofilament. In this paper we introduce the use of disulfide bond cross-linking to probe the fold within the core and the packing interactions between beta-sheets. In one approach, amyloid fibrils grown under reducing conditions from each of three double cysteine mutants (17/34, 17/35, and 17/36) of the Abeta(1-40) sequence were subjected to oxidizing conditions. Of these three mutants, only the Leu17Cys/Leu34Cys peptide could be cross-linked efficiently while resident in fibrils. In another approach, double Cys mutants were cross-linked as monomers before aggregation, and the resulting fibrils were assessed for stability, antibody binding, dye binding, and cross-seeding efficiency. Here too, fibrils from the 17/34 double Cys mutant most closely resemble wild-type Abeta(1-40) fibrils. These data support models of the Abeta fibril in which the Leu17 and Leu34 side chains of the same peptide pack against each other at the beta-sheet interface within the amyloid core. Related cross-linking strategies may reveal longer range spatial relationships. The ability of the cross-linked 17/35 double Cys mutant Abeta to also make amyloid fibrils illustrates a remarkable plasticity of the amyloid structure and suggests a structural mechanism for the generation of conformational variants of amyloid.     

Companion cell-specific inhibition of the potato sucrose transporter SUT1

In many plants, translocation of sucrose from mesnsophyll to phloem for long-distance transport is carrier-mediated. The sucrose H⁺-symporter gene SUT1 from potato is expressed at high levels in the phloem of mature, exporting leaves and at lower levels in other organs. Inhibition of SUT1 by expression of an antisense gene in companion cells under control of the rolC promoter leads to accumulation of high amounts of soluble and insoluble carbohydrates in leaves and inhibition of photosynthesis. The distribution of in situ localized starch does not correspond with areas of reduced photosynthesis as shown by fluorescence imaging. Dissection of antisense effects on sink and source organs by reciprocal grafts shows that inhibition of transporter gene expression in leaves is sufficient to produce chlorosis in leaves and reduced tuber yield. In contrast to the arrest of plasmodesmal development found in plants that express yeast invertase in the apoplast, in mature leaves of sucrose transporter antisense plants plasmodesmata are branched and have median cavities. These data strongly support an apoplastic mode of phloem loading in potato, in which the sucrose transporter located at the plasma membrane of the sieve element/companion cell complex represents the primary route for sugar uptake into the long-distance translocation pathway.     

Aging exacerbates damage and delays repair of alveolar epithelia following influenza viral pneumonia

Background

Influenza virus infection causes significantly higher levels of morbidity and mortality in the elderly. Studies have shown that impaired immunity in the elderly contributes to the increased susceptibility to influenza virus infection, however, how aging affects the lung tissue damage and repair has not been completely elucidated.

Methods

Aged (16–18 months old) and young (2–3 months old) mice were infected with influenza virus intratracheally. Body weight and mortality were monitored. Different days after infection, lung sections were stained to estimate the overall lung tissue damage and for club cells, pro-SPC⁺ bronchiolar epithelial cells, alveolar type I and II cells to quantify their frequencies using automated image analysis algorithms.

Results

Following influenza infection, aged mice lose more weight and die from otherwise sub-lethal influenza infection in young mice. Although there is no difference in damage and regeneration of club cells between the young and the aged mice, damage to alveolar type I and II cells (AT1s and AT2s) is exacerbated, and regeneration of AT2s and their precursors (pro-SPC-positive bronchiolar epithelial cells) is significantly delayed in the aged mice. We further show that oseltamivir treatment reduces virus load and lung damage, and promotes pulmonary recovery from infection in the aged mice.

Conclusions

These findings show that aging increases susceptibility of the distal lung epithelium to influenza infection and delays the emergence of pro-SPC positive progenitor cells during the repair process. Our findings also shed light on possible approaches to enhance the clinical management of severe influenza pneumonia in the elderly.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0116-z) contains supplementary material, which is available to authorized users.