Comparative analysis of inverted repeats of polypod fern (Polypodiales) plastomes reveals two hypervariable regions

Background

Ferns are large and underexplored group of vascular plants (~ 11 thousands species). The genomic data available by now include low coverage nuclear genomes sequences and partial sequences of mitochondrial genomes for six species and several plastid genomes.

Results

We characterized plastid genomes of three species of Dryopteris, which is one of the largest fern genera, using sequencing of chloroplast DNA enriched samples and performed comparative analysis with available plastomes of Polypodiales, the most species-rich group of ferns. We also sequenced the plastome of Adianthum hispidulum (Pteridaceae). Unexpectedly, we found high variability in the IR region, including duplication of rrn16 in D. blanfordii, complete loss of trnI-GAU in D. filix-mas, its pseudogenization due to the loss of an exon in D. blanfordii. Analysis of previously reported plastomes of Polypodiales demonstrated that Woodwardia unigemmata and Lepisorus clathratus have unusual insertions in the IR region. The sequence of these inserted regions has high similarity to several LSC fragments of ferns outside of Polypodiales and to spacer between tRNA-CGA and tRNA-TTT genes of mitochondrial genome of Asplenium nidus. We suggest that this reflects the ancient DNA transfer from mitochondrial to plastid genome occurred in a common ancestor of ferns. We determined the marked conservation of gene content and relative evolution rate of genes and intergenic spacers in the IRs of Polypodiales. Faster evolution of the four intergenic regions had been demonstrated (trnA- orf42, rrn16-rps12, rps7-psbA and ycf2-trnN).

Conclusions

IRs of Polypodiales plastomes are dynamic, driven by such events as gene loss, duplication and putative lateral transfer from mitochondria.

     

Selection of Peptide Mimics of HIV-1 Epitope Recognized by Neutralizing Antibody VRC01

The ability to induce anti-HIV-1 antibodies that can neutralize a broad spectrum of viral isolates from different subtypes seems to be a key requirement for development of an effective HIV-1 vaccine. The epitopes recognized by the most potent broadly neutralizing antibodies that have been characterized are largely discontinuous. Mimetics of such conformational epitopes could be potentially used as components of a synthetic immunogen that can elicit neutralizing antibodies. Here we used phage display technology to identify peptide motifs that mimic the epitope recognized by monoclonal antibody VRC01, which is able to neutralize up to 91% of circulating primary isolates. Three rounds of biopanning were performed against 2 different phage peptide libraries for this purpose. The binding specificity of selected phage clones to monoclonal antibody VRC01 was estimated using dot blot analysis. The putative peptide mimics exposed on the surface of selected phages were analyzed for conformational and linear homology to the surface of HIV-1 gp120 fragment using computational analysis. Corresponding peptides were synthesized and checked for their ability to interfere with neutralization activity of VRC01 in a competitive inhibition assay. One of the most common peptides selected from 12-mer phage library was found to partially mimic a CD4-binding loop fragment, whereas none of the circular C7C-mer peptides was able to mimic any HIV-1 domains. However, peptides identified from both the 12-mer and C7C-mer peptide libraries showed rescue of HIV-1 infectivity in the competitive inhibition assay. The identification of epitope mimics may lead to novel immunogens capable of inducing broadly reactive neutralizing antibodies.     

Barnacle settlement and the adhesion of protein and diatom microfouling to xerogel films with varying surface energy and water wettability

Previous work has shown that organosilica-based xerogels have the potential to control biofouling. In this study, modifications of chemistry were investigated with respect to their resistance to marine slimes and to settlement of barnacle cyprids. Adhesion force measurements of bovine serum albumin (BSA)-coated atomic force microscopy (AFM) tips to xerogel surfaces prepared from aminopropylsilyl-, fluorocarbonsilyl-, and hydrocarbonsilyl-containing precursors, indicated that adhesion was significantly less on the xerogel surfaces in comparison to a poly(dimethylsiloxane) elastomer (PDMSE) standard. The strength of adhesion of BSA on the xerogels was highest on surfaces with the highest and the lowest critical surface tensions, γ_C and surface energies, γ_S, and duplicated the ‘Baier curve’. The attachment to and removal of cells of the diatom Navicula perminuta from a similar series of xerogel surfaces were examined. Initial attachment of cells was comparable on all of the xerogel surfaces, but the percentage removal of attached cells by hydrodynamic shear stress increased with γ_C and increased wettability as measured by the static water contact angle, θ_Ws, of the xerogel surfaces. The percentage removal of cells of Navicula was linearly correlated with both properties (R ² = 0.74 for percentage removal as a function of θ_Ws and R ² = 0.69 for percentage removal as a function of γ_C). Several of the aminopropylsilyl-containing xerogels showed significantly greater removal of Navicula compared to a PDMSE standard. Cypris larvae of the barnacle B. amphitrite showed preferred settlement on hydrophilic/higher energy surfaces. Settlement was linearly correlated with θ_Ws (R ² = 0.84) and γ_C (R ² = 0.84). Hydrophilic xerogels should prove useful as coatings for boats in regions where fouling is dominated by microfouling (protein and diatom slimes).     

Identification of variation in adaptively important traits and genome-wide analysis of trait-marker associations in Triticum monococcum

Einkorn wheat Triticum monococcum (2n=2x=14, A(m)A(m)) is one of the earliest domesticated crops. However, it was abandoned for cultivation before the Bronze Age and has infrequently been used in wheat breeding. Little is known about the genetic variation in adaptively important biological traits in T. monococcum. A collection of 30 accessions of diverse geographic origins were characterized for phenotypic variation in various agro-morphological traits including grain storage proteins and endosperm texture, nucleotide-binding site (NBS) domain profiles of resistance (R) genes and resistance gene analogues (RGAs), and germination under salt and drought stresses. Forty-six SSR (single sequence repeat) markers from bread wheat (T. aestivum, 2n=6x=42, AABBDD) A genome were used to establish trait-marker associations using linear mixed models. Multiple significant associations were identified, some of which were on chromosomal regions containing previously known genetic loci. It is concluded that T. monococcum possesses large genetic diversity in multiple traits. The findings also indicate that the efficiency of association mapping is much higher in T. monococcum than in other plant species. The use of T. monococcum as a reference species for wheat functional genomics is discussed.     

Mechanosensitive channel activity and F-actin organization in cholesterol-depleted human leukaemia cells

This study focuses on the functional role of cellular cholesterol in the regulation of mechanosensitive cation channels activated by stretch in human leukaemia K562 cells. The patch-clamp method was employed to examine the effect of methyl-beta-cyclodextrin (MbetaCD), a synthetic cholesterol-sequestering agent, on stretch-activated single currents. We found that cholesterol-depleting treatment with MbetaCD resulted in a suppression of the activity of mechanosensitive channels without a change in the unitary conductance. The probability that the channel was open significantly decreased after treatment with MbetaCD. Fluorescent microscopy revealed F-actin reorganization, possibly involving actin assembly, after incubation of the cells with MbetaCD. We suggest that suppression of mechanosensitive channel activation in cholesterol-depleted leukaemia cells is due to F-actin rearrangement, presumably induced by lipid raft destruction. Our observations are consistent with the notion that stretch-activated cation channels in eukaryotic cells are regulated by the membrane-cytoskeleton complex rather than by tension developed purely in the lipid bilayer.     

Nickel and zinc effects,accumulation and distribution in ruderal plants Lepidium ruderale and Capsella bursa-pastoris

Ruderal plants can grow in polluted areas, but little is known about heavy metal accumulation and distribution in them. Here Ni and Zn accumulation, distribution and effects were investigated in Lepidium ruderale and Capsella bursa-pastoris grown at 5–30 µM Ni(NO₃)₂ or 10–80 µM Zn(NO₃)₂. Metal contents were measured by flame atomic absorption spectrophotometry and tissue distribution of metals was studied histochemically. Ni was more toxic than Zn for both plants. When metal-induced growth-inhibiting effects were compared at various metal concentrations in solution, L. ruderale was more tolerant to Ni, whereas C. bursa-pastoris to Zn. However, when compared at similar Zn or Ni contents in roots, root growth of C. bursa-pastoris was more tolerant than that of L. ruderale. On the contrary, at similar Zn or Ni contents in shoots, shoot growth of L. ruderale was more tolerant. Both plants are excluders maintaining low metal levels in shoots. In roots, Ni located in protoplasts while Zn was also detected in cell walls. Metal accumulation in root apices resulted in growth inhibition. Ni accumulation in root cortex constrained metal translocation into central cylinder and then to shoots, where it located only in conductive tissues and epidermis, particularly in leaf trichomes of C. bursa-pastoris. Zn was translocated to shoots more actively and distributed in all shoot tissues, being accumulated in leaf vascular bundles and epidermis. To conclude, these patterns of Ni and Zn distribution are aimed at metal sequestration in roots and leaf epidermis, thus keeping mesophyll from metal penetration and pigment degradation.     

Identification of cell type–specific correlations between ERK activity and cell viability upon treatment with ERK1/2 inhibitors

Increased MAPK signaling is a hallmark of various cancers and is a central regulator of cell survival. Direct ERK1/2 inhibition is considered a promising approach to avoid ERK1/2 reactivation caused by upstream kinases BRAF, MEK1/2, and KRAS, as well as by receptor tyrosine kinase inhibitors, but the dynamics and selectivity of ERK1/2 inhibitors are much less studied compared with BRAF or MEK inhibitors. Using ERK1/2 and downstream kinase ELK1 reporter cell lines of lung cancer (H1299; NRAS^Q61K), colon cancer (HCT-116; KRAS^G13D), neuroblastoma (SH-SY5Y), and leukemia (U937), we examined the relationship between ERK inhibition and drug-induced toxicity for five ERK inhibitors: SCH772984, ravoxertinib, LY3214996, ulixertinib, and VX-11e, as well as one MEK inhibitor, PD0325901. Comparing cell viability and ERK inhibition revealed different ERK dependencies for these cell lines. We identify several drugs, such as SCH772984 and VX-11e, which induce excessive toxicity not directly related to ERK1/2 inhibition in specific cell lines. We also show that PD0325901, LY3214996, and ulixertinib are prone to ERK1/2 reactivation over time. We distinguished two types of ERK1/2 reactivation: the first could be reversed by adding a fresh dose of inhibitors, while the second persists even after additional treatments. We also showed that cells that became resistant to the MEK1/2 inhibitor PD0325901 due to ERK1/2 reactivation remained sensitive to ERK1/2 inhibitor ulixertinib. Our data indicate that correlation of ERK inhibition with drug-induced toxicity in multiple cell lines may help to find more selective and effective ERK1/2 inhibitors.