Septal localization of the Mycobacterium tuberculosis MtrB sensor kinase promotes MtrA regulon expression

The mechanisms responsible for activation of the MtrAB two-component regulatory signal transduction system, which includes sensor kinase MtrB and response regulator MtrA, are unknown. Here, we show that an MtrB-GFP fusion protein localized to the cell membrane, the septa, and the poles in Mycobacterium tuberculosis and Mycobacterium smegmatis. This localization was independent of MtrB phosphorylation status but dependent upon the assembly of FtsZ, the initiator of cell division. The M. smegmatis mtrB mutant was filamentous, defective for cell division, and contained lysozyme-sensitive cell walls. The mtrB phenotype was complemented by either production of MtrB protein competent for phosphorylation or overproduction of MtrA(Y102C) and MtrA(D13A) mutant proteins exhibiting altered phosphorylation potential, indicating that either MtrB phosphorylation or MtrB independent expression of MtrA regulon genes, including those involved in cell wall processing, are necessary for regulated cell division. In partial support of this observation, we found that the essential cell wall hydrolase ripA is an MtrA target and that the expression of bona fide MtrA targets ripA, fbpB, and dnaA were compromised in the mtrB mutant and partially rescued upon MtrA(Y102C) and MtrA(D13A) overproduction. MtrB septal assembly was compromised upon FtsZ depletion and exposure of cells to mitomycin C, a DNA damaging agent, which interferes with FtsZ ring assembly. Expression of MtrA targets was also compromised under the above conditions, indicating that MtrB septal localization and MtrA regulon expression are linked. We propose that MtrB septal association is a necessary feature of MtrB activation that promotes MtrA phosphorylation and MtrA regulon expression.     

Optimization of culture conditions of Arnica montana L.: effects of mycorrhizal fungi and competing plants

Arnica montana is a rare plant that needs special protection because of its intensive harvesting for medicinal purposes. The present work was aimed at finding optimal culture conditions for Arnica plants in order to enable their successful reintroduction into their natural stands. Plants were cultivated under controlled greenhouse conditions on substrata with different nitrogen (N) concentration. As Arnica is always colonized by arbuscular mycorrhizal fungi (AMF) in nature, a fact that has been overlooked in other similar projects, we, here, applied and tested different inocula. We found that they differed in their effectiveness, both in establishing symbiosis, assessed by the colonization parameters, and in improving the performance of Arnica, evaluated by the photosynthetic parameters derived from the fluorescence transients (JIP-test), with the inocula containing G. intraradices or composed of several Glomus strains being the most effective. The comparison was possible only on substrata with medium N, since high N did not permit the formation of mycorrhiza, while at low N, few nonmycorrhizal plants survived until the measurements and mycorrhizal plants, which were well growing, exhibited a high heterogeneity. Analysis of secondary metabolites showed clearly that mycorrhization was associated with increased concentrations of phenolic acids in roots. For some of the inocula used, a tendency for increase of the level of phenolic acids in shoots and of sesquiterpene lactones, both in roots and in shoots, was also observed. We also studied the interactions between A. montana and Dactylis glomerata, known to compete with Arnica under field conditions. When specimens from both species were cultured together, there was no effect on D. glomerata, but Arnica could retain a photosynthetic performance that permitted survivability only in the presence of AMF; without AMF, the photosynthetic performance was lower, and the plants were eventually totally outcompeted.     

Quantitative modeling of Arabidopsis development

We present an empirical model of Arabidopsis (Arabidopsis thaliana), intended as a framework for quantitative understanding of plant development. The model simulates and realistically visualizes development of aerial parts of the plant from seedling to maturity. It integrates thousands of measurements, taken from several plants at frequent time intervals. These data are used to infer growth curves, allometric relations, and progression of shapes over time, which are incorporated into the final three-dimensional model. Through the process of model construction, we identify the key attributes required to characterize the development of Arabidopsis plant form over time. The model provides a basis for integrating experimental data and constructing mechanistic models.     

Molstack: A platform for interactive presentations of electron density and cryo‐EM maps and their interpretations

In the Special Issue on Tools for Protein Science in 2018, we presented Molstack: a concept of a cloud‐based platform for sharing electron density maps and their interpretations. Molstack is a web platform that allows the interactive visualization of density maps through the simultaneous presentation of multiple datasets and models in a way that allows for easy pairwise comparison. We anticipated that the users of this conceptually simple platform would find many different uses for their projects, and we were not mistaken. We have observed researchers use Molstack to present experimental evidence for their models in the form of electron density maps, omit maps, and anomalous difference density maps. Users also employed Molstack to present alternative interpretations of densities, including rerefinements and speculative interpretations. While we anticipated these types of projects to be the main use cases, we were pleased to see Molstack used to display superpositions of different models, as a tool for story‐driven presentations, and for collaboration as well. Here, we present developments in the platform that were driven by user feedback, highlight several cases that used Molstack to enhance the publication, and discuss possible directions for the platform.     

NAR Breakthrough Article: Genome-wide analyses of LINE–LINE-mediated nonallelic homologous recombination

Nonallelic homologous recombination (NAHR), occurring between low-copy repeats (LCRs) >10 kb in size and sharing >97% DNA sequence identity, is responsible for the majority of recurrent genomic rearrangements in the human genome. Recent studies have shown that transposable elements (TEs) can also mediate recurrent deletions and translocations, indicating the features of substrates that mediate NAHR may be significantly less stringent than previously believed. Using >4 kb length and >95% sequence identity criteria, we analyzed of the genome-wide distribution of long interspersed element (LINE) retrotransposon and their potential to mediate NAHR. We identified 17 005 directly oriented LINE pairs located <10 Mbp from each other as potential NAHR substrates, placing 82.8% of the human genome at risk of LINE–LINE-mediated instability. Cross-referencing these regions with CNVs in the Baylor College of Medicine clinical chromosomal microarray database of 36 285 patients, we identified 516 CNVs potentially mediated by LINEs. Using long-range PCR of five different genomic regions in a total of 44 patients, we confirmed that the CNV breakpoints in each patient map within the LINE elements. To additionally assess the scale of LINE–LINE/NAHR phenomenon in the human genome, we tested DNA samples from six healthy individuals on a custom aCGH microarray targeting LINE elements predicted to mediate CNVs and identified 25 LINE–LINE rearrangements. Our data indicate that LINE–LINE-mediated NAHR is widespread and under-recognized, and is an important mechanism of structural rearrangement contributing to human genomic variability.     

A Novel Short Isoform of Cytosolic PSD-95 Interactor (Cypin) Regulates Neuronal Development

The guanine deaminase cypin (cytosolic PSD-95 interactor) binds to PSD-95 (postsynaptic density protein 95) and regulates dendrite branching by promoting microtubule polymerization. Here, we identify a novel short isoform of cypin, termed cypinS, which is expressed in mouse and human, but not rat, tissues. Cypin and cypinS mRNA and protein levels peak at P7 and P14 in the mouse brain, suggesting a role for these isoforms during development. Interestingly, although cypinS lacks guanine deaminase activity, overexpression of cypinS increases dendrite branching. This increase occurs further away from soma than do increases resulting from overexpression of cypin. In contrast, overexpression of cypin, but not cypinS, decreases dendritic spine density and maturity. This suggests that changes to spines, but not to dendrites, may be dependent on guanine deaminase activity. Furthermore, overexpression of either cypin or cypinS increases miniature excitatory postsynaptic current (mEPSC) frequency, pointing to a presynaptic role for both isoforms. Interestingly, overexpression of cypinS results in a significantly greater increase in frequency than does overexpression of cypin. Thus, cypin and cypinS play distinct roles in neuronal development.     

Different procedures of diphenyleneiodonium chloride addition affect neutrophil extracellular trap formation

A unique strategy, in which invading microorganisms are being caught in web-like structures composed mainly of DNA, involves a recently described phenomenon called NETosis. This process seems to be related to the production of reactive oxygen species (ROS). In our study, the influence of diphenyleneiodonium chloride (DPI), which diminishes ROS production, was assessed in the context of neutrophil extracellular trap (NET) release. According to protocol, two distinguished procedures were compared, the first one involving DPI elimination from sample before cell activation and the second one proceeding without the step of inhibitor washout. The kinetics of DNA release was monitored by fluorometric assay, and NET formation was observed by fluorescent microscopy. The addition of DPI to the sample led to a reduction of extracellular DNA release. The strongest inhibition was noticed after treatment with 10 μM DPI, which was removed from medium before stimulation with phorbol-12-myristate-13-acetate (PMA). Our findings confirmed that DPI is able to block NET creation. However, the addition of DPI together with PMA or the addition of inhibitor initially and then washing it out before stimulation resulted in different levels of NET formation. Finally, DPI that remained in the system induced specific morphological changes in the neutrophils' nuclei that was not observed in the DPI washed out from sample.