CRISPR/Cas9 Mutagenesis through Introducing a Nanoparticle Complex Made of a Cationic Polymer and Nucleic Acids into Maize Protoplasts.

Presently, targeted gene mutagenesis attracts increasing attention both in plant research and crop improvement. In these approaches, successes are largely dependent on the efficiency of the delivery of gene editing components into plant cells. Here, we report the optimization of the cationic polymer poly(2-hydroxypropylene imine) (PHPI)-mediated delivery of plasmid DNAs, or single-stranded oligonucleotides labelled with Cyanine3 (Cy3) or 6-Carboxyfluorescein (6-FAM)-fluorescent dyes into maize protoplasts. Co-delivery of the GFP-expressing plasmid and the Cy3-conjugated oligonucleotides has resulted in the cytoplasmic and nuclear accumulation of the green fluorescent protein and a preferential nuclear localization of oligonucleotides. We show the application of nanoparticle complexes, i.e., "polyplexes" that comprise cationic polymers and nucleic acids, for CRISPR/Cas9 editing of maize cells. Knocking out the functional EGFP gene in transgenic maize protoplasts was achieved through the co-delivery of plasmids encoding components of the editing factors Cas9 (pFGC-pcoCas9) and gRNA (pZmU3-gRNA) after complexing with a cationic polymer (PHPI). Several edited microcalli were identified based on the lack of a GFP fluorescence signal. Multi-base and single-base deletions in the EGFP gene were confirmed using Sanger sequencing. The presented results support the use of the PHPI cationic polymer in plant protoplast-mediated genome editing approaches.

Insights into the molecular architecture of the 26S proteasome.

Cryo-electron microscopy in conjunction with advanced image analysis was used to analyze the structure of the 26S proteasome and to elucidate its variable features. We have been able to outline the boundaries of the ATPase module in the "base" part of the regulatory complex that can vary in its position and orientation relative to the 20S core particle. This variation is consistent with the "wobbling" model that was previously proposed to explain the role of the regulatory complex in opening the gate in the alpha-rings of the core particle. In addition, a variable mass near the mouth of the ATPase ring has been identified as Rpn10, a multiubiquitin receptor, by correlating the electron microscopy data with quantitative mass spectrometry.

In vitro FT-IR study of the effects of hydrogen peroxide on superficial tooth enamel.

OBJECTIVES: The aim of the present study was to determine the alteration in human enamel after hydrogen peroxide treatment using FT-IR spectroscopy. It is hypothesized that infrared spectroscopy is capable of showing alterations in human enamel after peroxide treatment and the alteration in enamel is proportional to peroxide concentration. METHODS: The effects of 10, 20 and 30% hydrogen peroxide solutions on human enamel were tested. Thirty non-carious human teeth, extracted for periodontal reasons, were used in this study. They were divided into 3 groups of 10, according to the peroxide concentration, sectioned, and the specimens were embedded in resin for infrared spectroscopic analysis. The total treatment time was 120 min. Spectra of the specimens were taken before treatment and 30, 60 and 120 min after it. Another spectrum was taken in a week. RESULTS: Infrared spectroscopic analysis showed two distinct bands (biological PO(4)nu1 and nu2) that were capable of describing the alterations in enamel structure. On comparing the infrared spectra of non-treated and treated specimens, structural changes were detected in the superficial enamel. The alteration in enamel was proportional to treatment time and hydrogen peroxide concentration. Higher concentration and longer treatment time resulted in more severe alterations. The numerical analysis of the spectra revealed that on using concentrated hydrogen peroxide solutions the alterations of the IR spectra were more pronounced. The spectra taken in 1 week after treatment did not show spontaneous reversibility in enamel structure. CONCLUSION: At-home and in-office peroxide-containing bleaching agents are capable of causing alteration in enamel at low and high concentrations as well. According to the results of this study it is recommended to perform tooth whitening using low concentration of hydrogen and/or carbamide peroxide, and shorten treatment time to reduce the possible destruction but reach the required change in color.

Thermobifida cellulolytica sp. nov., a novel lignocellulose-decomposing actinomycete.

Four actinomycete strains, isolated from the overheated region of manure compost, were assigned to the genus Thermobifida on the basis of morphological, physiological and biochemical characteristics. All strains produced single, ovoid, heat-sensitive spores on dichotomically branched aerial hyphae. On the basis of chemotaxonomic traits, these isolates showed strong affinity towards members of the genus Thermobifida. Cell-wall analysis revealed the presence of meso-diaminopimelic acid, but no other characteristic amino acids or sugars in the murein (cell wall type III). According to polar lipid analysis, all strains showed PL II-type phospholipid composition; phosphatidylethanolamine and glycolipid were detected together with some unidentified phospholipids. The isoprenoid quinone composition of the new isolates differed slightly from that of the other two Thermobifida species described thus far. The partial 16S rDNA sequence similarity of the four strains reached 99.8-100%, whereas a nearly complete 16S rDNA sequence of TB100T, the representative strain of this collection, showed only 97.4 and 97.8% similarity to the corresponding rDNA sequences of the type strains of Thermobifida fusca and Thermobifida alba, respectively. These four isolates constituted a homogeneous group with levels of DNA-DNA homology ranging from 94.6 to 99.1%. The DNA-DNA relative homology values of strain TB100T to Thermobifida fusca ATCC 27730T and Thermobifida alba DSM 43795T were 48.1 and 57%, respectively. On the basis of phenotypic, chemotaxonomic and genotypic data, the strains are assigned to a new species within the genus Thermobifida under the name Thermobifida cellulolytica sp. nov. The type strain is TB100T (= DSM 44535T = NCAIM B01997T).