Phylodynamic Inference of Bacterial Outbreak Parameters Using Nanopore Sequencing

Nanopore sequencing and phylodynamic modeling have been used to reconstruct the transmission dynamics of viral epidemics, but their application to bacterial pathogens has remained challenging. Cost-effective bacterial genome sequencing and variant calling on nanopore platforms would greatly enhance surveillance and outbreak response in communities without access to sequencing infrastructure. Here, we adapt random forest models for single nucleotide polymorphism (SNP) polishing developed by Sanderson and colleagues (2020. High precision Neisseria gonorrhoeae variant and antimicrobial resistance calling from metagenomic nanopore sequencing. Genome Res. 30(9):1354–1363) to estimate divergence and effective reproduction numbers (R_e) of two methicillin-resistant Staphylococcus aureus (MRSA) outbreaks from remote communities in Far North Queensland and Papua New Guinea (PNG; n = 159). Successive barcoded panels of S. aureus isolates (2 × 12 per MinION) sequenced at low coverage (>5× to 10×) provided sufficient data to accurately infer genotypes with high recall when compared with Illumina references. Random forest models achieved high resolution on ST93 outbreak sequence types (>90% accuracy and precision) and enabled phylodynamic inference of epidemiological parameters using birth–death skyline models. Our method reproduced phylogenetic topology, origin of the outbreaks, and indications of epidemic growth (R_e > 1). Nextflow pipelines implement SNP polisher training, evaluation, and outbreak alignments, enabling reconstruction of within-lineage transmission dynamics for infection control of bacterial disease outbreaks on portable nanopore platforms. Our study shows that nanopore technology can be used for bacterial outbreak reconstruction at competitive costs, providing opportunities for infection control in hospitals and communities without access to sequencing infrastructure, such as in remote northern Australia and PNG.     

Morphological events in cultures of mechanically isolated maize mcirospores

Summary In tis androgenic response, maize is considered to be a recalcitrant plant. We used mechanically isolated microspores of maize genotype A18 to establish a responsive microspore culture of maize. Morphological events occurring during the first days of maize androgenesis in a microspore culture were observed and described, and some morphological markers for distinguishing between embryogenic microspores and nonembryogenic microspores were identified. It was found that the enlargement of microspores during the first days in culture and the ‘star-like’ organization of the cytoplasm inside the microspore are connected with reprogramming of the developmental pathway in maize microspores. Some differences were also found in the surface wall architecture of embryogenic microspores. Fertile plants were successfully recovered from microspore-originated structures.     

Calreticulin mRNA and protein are localized to protein bodies in storage maize callus cells

Maize callus cells possess numerous protein bodies which develop as sub-compartments of the endoplasmic reticulum. We localized maize calreticulin mRNAs and protein in maize callus cells using in situ hybridization and immunocytochemistry. Calreticulin mRNAs were selectively targeted to the endoplasmic reticulum (ER) subdomains surrounding protein bodies. Profilin mRNAs, used as a positive control for in situ hybridization experiments, showed distinct and rather diffuse localization pattern. Using both, immunofluorescence and immunogold electron microscopy localization techniques, calreticulin was found to be enriched around and within protein bodies in maize callus storage cells. As a positive control for reticuloplasmins, HDEL antibody revealed labelling of protein bodies and of the nuclear envelope. The identity of protein bodies was confirmed by specific binding of an α zein antibody. These data suggest that calreticulin mRNA is targeted towards protein body forming subdomains of the ER, and that calreticulin is localized and enriched in these protein bodies. The possibility that calreticulin plays an important role in zein retention within the ER and/or its assembly and packaging into protein bodies during protein body biogenesis in maize callus is discussed.     

喀斯特11种典型生态恢复树种凋落叶分解及其对土壤碳排放的激发效应

旨在探究喀斯特地区退化生态系统植被恢复树种凋落叶分解过程及其对土壤碳排放的激发效应,为选择合适的树种进行植被恢复提供数据支持。以中国林科院热带林业实验中心大青山石山树木园11种适应性强、耐干旱贫瘠的优良石山树种为研究对象,利用¹³C自然丰度法区分凋落叶和土壤来源CO₂并量化土壤激发效应,比较不同生态恢复树种凋落叶分解及其激发效应的差异,探讨凋落物分解及其激发效应与凋落物性状之间的关联。结果表明:(1)11个生态恢复树种凋落叶在碳相关化学性质(水溶性碳、半纤维素和单宁含量等)、养分含量(磷和镁含量等)及化学计量特征(碳磷比和氮磷比)等方面均表现出较高程度变异。(2)不同生态恢复树种凋落叶分解及其诱导的土壤激发效应具有极显著差异(P<0.001);在整个培养实验期间,11个生态恢复树种凋落叶平均分解了35.3%,其中海南椴分解最快,达到50%,而青冈栎分解最慢,仅分解16.5%。(3)总体上看,凋落叶处理的土壤呼吸速率(5.1 mg C kg^-1 土壤 d^-1)是对照土壤呼吸速率(2.3 mg C kg^-1土壤d^-1)的2.2倍,凋落叶添加显著促进土壤有机碳分解,平均达到37.6%;其中海南椴、割舌树和任豆凋落叶输入则抑制土壤有机碳分解(抑制程度分别为-13.2%、-6.9%和-22.5%),产生负激发效应。(4)凋落叶分解与非结构性碳(r=0.63,P=0.04)和水溶性碳(r=0.91,P<0.001)呈显著正相关,与叶干物质含量(r=0.64,P=0.03)、纤维素(r=0.62,P=0.04)和锰含量(r=-0.63,P=0.04)呈显著负相关。多元回归分析结果表明,水溶性碳、钾和钙含量相结合可以解释生态恢复树种凋落叶分解变异的98%;然而,凋落叶性状与土壤激发效应强度之间并没有显著相关性。从土壤养分归还角度考虑,喀斯特退化生态系统恢复树种可以选择光皮梾木、海南椴、顶果木和降香黄檀等凋落叶分解较快的树种,以促进土壤养分循环和植被恢复;另一方面,从土壤碳固持角度来看,海南椴、任豆和割舌树等凋落叶输入会抑制土壤有机碳分解,从而有利于提高退化生态系统土壤碳封存能力。     

Role of some members of enterobacteriacae family in maintaining and spreading of the transferable polyresistance

The relation of the R₆ episomal factor to the main representatives of the Enterobacteriacae family was studied. The best recipients of the R factor were found the derivatives ofEscherichia coli K-12, the worst ones Salmonellae, Citrobacters and Aerobacters. The highest phenotypical expression was attained in the resistance to sulfonamides and kanamycin (more than 1000 ug/ml), the lowest one in the resistance to streptomycine (20–100 ug/ml). The highest segregation of the R₆ factor was observed inSalmonella typhi andSalmonella typhimurium. In other strains the segregation was low. Besides the R^? cells eight types of segregants, none of them conspicuously prevalent, were observed. All types of segregants retained the ability to transfer the remaining resistance factors. The course of segregation in broth and during experimental keratoconjunctivitis did not differ in all strains which could have been investigated under these conditions. Salmonellae, in which the highest segregation was demonstrated, probably cannot serve as a stable source of R factors.     

An evaluation of three pesticides: Piritione,supercypermethrin and metolachlor in transformation bioassays of BHK21 and hamster embryo cells

In a study of potential carcinogenicity of pesticides, Piritione, metolachlor (in the form of Dual and VUCHT 524) and Supercypermethrin (in the form of Supercypermethrin EC and Supercypermethrin TP) were assayed for induction of anchorage independent growth of BHK21 cells and morphological transformation of Syrian hamster embryo cells. The activity of these substances in both transformation assays was compared to the activity of the direct-acting ultimate carcinogen N-methyl-N-nitrosourea. In comparison to the very strong transforming activity of N-methyl-N-nitrosourea all pesticides tested with or without S9 fraction manifested a very weak, weak, medium or strong effect. The ability to induce anchorage independent growth was graded as follows: Dual < Supercypermethrin EC < Supercypermethrin TP Piritione < VUCHT 524. Results of Syrian hamster embryo cell transformation assay were very similar to the BKH21 transformation assay. VUCHT 524 strongly induced transformation whereas Dual was inactive. Piritione and Supercypermethrin EC and Supercypermethrin TP elicited a slight but significant positive response.     

Plant cell and biotechnology studies in <Emphasis Type="Italic">Linum usitatissimum</Emphasis> – a review

The species Linum usitatissimum (flax/linseed) has been the focus of a great deal of both basic and applied research effort in plant cell and biotechnology studies in recent years. In this review we consider applications of the techniques of plant biotechnology in this species under several distinct headings. Plant cell and tissue regeneration strategies and applications are discussed, and the applications of the techniques of somatic embryogenesis, protoplast isolation, culture and fusion and cell suspension cultures in this species are described. A major area of study is the use of anther and microspore culture where clear advantages to breeding programmes could be applied. In addition, embryo and ovary culture studies have resulted in significant findings. The more recent technologies of gene transfer and expression by genetic transformation are reviewed, and a section on strategies for improvements in technological quality is also included. Finally we propose conclusions and future prospects for this ancient, but still highly relevant crop.     

ER disruption and GFP degradation during non-regenerable transformation of flax with Agrobacterium tumefaciens

Flax is considered as plant species susceptible to Agrobacterium-mediated genetic transformation. In this study, stability of flax transformation by Agrobacterium rhizogenes versus Agrobacterium tumefaciens was tested by using combined selection for antibiotic resistance and visual selection of green fluorescent protein (GFP)-fusion reporter targeted to the endoplasmic reticulum (ER). Transformation with A. rhizogenes was stable for over 2 years, whereas transformation by A. tumefaciens resulted in non-regenerable stable transformation which was restricted solely to transgenic callus and lasted only 6–8 weeks. However, shoots regenerated from this callus appeared to be non-transgenic. Importantly, callus and root cells stably transformed with A. rhizogenes showed typical regular organization and dynamics of ER as visualized by GFP-ER marker. On the other hand, callus cells transformed with A. tumefaciens showed disintegrated ER structure and impaired dynamics which was accompanied with developmental degradation of GFP. Consequently, shoots which regenerated from such callus were all non-transgenic. Possible reasons for this non-regenerable flax transformation by A. tumefaciens are discussed.