3D nanomechanical mapping of subcellular and sub-nuclear structures of living cells by multi-harmonic AFM with long-tip microcantilevers

Recent developments such as multi-harmonic Atomic Force Microscopy (AFM) techniques have enabled fast, quantitative mapping of nanomechanical properties of living cells. Due to their high spatiotemporal resolution, these methods provide new insights into changes of mechanical properties of subcellular structures due to disease or drug response. Here, we propose three new improvements to significantly improve the resolution, identification, and mechanical property quantification of sub-cellular and sub-nuclear structures using multi-harmonic AFM on living cells. First, microcantilever tips are streamlined using long-carbon tips to minimize long-range hydrodynamic interactions with the cell surface, to enhance the spatial resolution of nanomechanical maps and minimize hydrodynamic artifacts. Second, simultaneous Spinning Disk Confocal Microscopy (SDC) with live-cell fluorescent markers enables the unambiguous correlation between observed heterogeneities in nanomechanical maps with subcellular structures. Third, computational approaches are then used to estimate the mechanical properties of sub-nuclear structures. Results are demonstrated on living NIH 3T3 fibroblasts and breast cancer MDA-MB-231 cells, where properties of nucleoli, a deep intracellular structure, were assessed. The integrated approach opens the door to study the mechanobiology of sub-cellular structures during disease or drug response.

Nuclear Structure Evolution Reflected from Local Relations

The structure evolution of nuclei which are in connection with symmetry breaking is one of the important problems not only for nuclear structures, but also for astrophysics and the spectroscopy of exotic nuclei. Many physical quantities can provide useful information of a shell structure, such as nuclear masses and nuclear charge radii. This paper introduces three kinds of local relations, i.e., the NpNn scheme respectively for the quadrupole deformation parameter and the excitation energy of the first 2+, 4+, 6+ states, the (αN′n+N′p) relation for nuclear charge radii and α decay energies, and the so-called “nonpairing” relation for binding energies and nuclear charge radii. All these relations reflect the evolution of nuclear structures, involving shells, subshells, shape coexistence, phase transition and the Wigner effect. Some results from different models can be verified with each other.

Chloroplast Ultrastructure and Photosynthetic Response of the Dinoflagellate Akashiwo sanguinea Throughout Infection by Amoebophrya sp.

The endoparasitic dinoflagellate Amoebophrya infects a number of marine dinoflagellates, including toxic and harmful algal bloom-forming species. The parasite kills its host and has been proposed to be a determining factor in the demise of dinoflagellate blooms in restricted coastal waters. Previous studies have mainly focused on the occurrence, prevalence, and diversity of Amoebophrya, while the interactions between the parasite and its host have received limited attention. Herein, an Amoebophrya sp.-Akashiwo sanguinea co-culture was established from Chinese coastal waters, and morphological, physiological, and transcriptional changes throughout an infection cycle of the parasite were systemically studied. The parasitic dinoflagellate was very infectious, resulting in an infection rate up to 85.83% at a dinospore:host ratio of 10:1. Infected host cells died eventually and released approximately 370 dinospores/cell. The host nuclear structures were rapidly degraded by Amoebophrya infection, and the chloroplasts of parasitized host cells remained intact until the parasite filled the almost entire cell structure. Nevertheless, infected cultures showed sustained but lower levels of photosynthetic performance (∼64% of control cultures), and the photosynthesis-related genes were significantly down-regulated. These findings provide a better understanding of the biological basis of the complex parasite-host interactions, which will be helpful to further elucidate the ecological significance of parasitic dinoflagellates in marine ecosystems.

PIAS Factors from Rainbow Trout Control NF-κB- and STAT-Dependent Gene Expression

Four ‘protein inhibitors of activated STAT’ (PIAS) control STAT-dependent and NF-κB-dependent immune signalling in humans. The genome of rainbow trout (Oncorhynchus mykiss) contains eight pias genes, which encode at least 14 different pias transcripts that are differentially expressed in a tissue- and cell-specific manner. Pias1a2 was the most strongly expressed variant among the analysed pias genes in most tissues, while pias4a2 was commonly low or absent. Since the knock-out of Pias factors in salmonid CHSE cells using CRISPR/Cas9 technology failed, three structurally different Pias protein variants were selected for overexpression studies in CHSE-214 cells. All three factors quenched the basal activity of an NF-κB promoter in a dose-dependent fashion, while the activity of an Mx promoter remained unaffected. Nevertheless, all three overexpressed Pias variants from trout strongly reduced the transcript level of the antiviral Stat-dependent mx gene in ifnγ-expressing CHSE-214 cells. Unlike mx, the overexpressed Pias factors modulated the transcript levels of NF-κB-dependent immune genes (mainly il6, il10, ifna3, and stat4) in ifnγ-expressing CHSE-214 cells in different ways. This dissimilar modulation of expression may result from the physical cooperation of the Pias proteins from trout with differential sets of interacting factors bound to distinct nuclear structures, as reflected by the differential nuclear localisation of trout Pias factors. In conclusion, this study provides evidence for the multiplication of pias genes and their sub-functionalisation during salmonid evolution.

A Shift in Paradigms: Spatial Genomics Approaches to Reveal Single-Cell Principles of Genome Organization

The genome tridimensional (3D) organization and its role towards the regulation of key cell processes such as transcription is currently a main question in biology. Interphase chromosomes are spatially segregated into “territories,” epigenetically-defined large domains of chromatin that interact to form “compartments” with common transcriptional status, and insulator-flanked domains called “topologically associating domains” (TADs). Moreover, chromatin organizes around nuclear structures such as lamina, speckles, or the nucleolus to acquire a higher-order genome organization. Due to recent technological advances, the different hierarchies are being solved. Particularly, advances in microscopy technologies are shedding light on the genome structure at multiple levels. Intriguingly, more and more reports point to high variability and stochasticity at the single-cell level. However, the functional consequences of such variability in genome conformation are still unsolved. Here, I will discuss the implication of the cell-to-cell heterogeneity at the different scales in the context of newly developed imaging approaches, particularly multiplexed Fluorescence in situ hybridization methods that enabled “chromatin tracing.” Extensions of these methods are now combining spatial information of dozens to thousands of genomic loci with the localization of nuclear features such as the nucleolus, nuclear speckles, or even histone modifications, creating the fast-moving field of “spatial genomics.” As our view of genome organization shifts the focus from ensemble to single-cell, new insights to fundamental questions begin to emerge.

Sequencing Micronuclei Reveals the Landscape of Chromosomal Instability

Genome instability (GIN) is a main contributing factor to congenital and somatic diseases, but its sporadic occurrence in individual cell cycles makes it difficult to study mechanistically. One profound manifestation of GIN is the formation of micronuclei (MN), the engulfment of chromosomes or chromosome fragments in their own nuclear structures separate from the main nucleus. Here, we developed MN-seq, an approach for sequencing the DNA contained within micronuclei. We applied MN-seq to mice with mutations in Mcm4 and Rad9a, which disrupt DNA replication, repair, and damage responses. Data analysis and simulations show that centromere presence, fragment length, and a heterogenous landscape of chromosomal fragility all contribute to the patterns of DNA present within MN. In particular, we show that long genes, but also gene-poor regions, are associated with chromosome breaks that lead to the enrichment of particular genomic sequences in MN, in a genetic background-specific manner. Finally, we introduce single-cell micronucleus sequencing (scMN-seq), an approach to sequence the DNA present in MN of individual cells. Together, sequencing micronuclei provides a systematic approach for studying GIN and reveals novel molecular associations with chromosome breakage and segregation.

Model of Translation of International Legal Discourse: to the Problem of Applying the Transformation Model and the Model of the Reference Set of Features

The article focuses on analyzes of the translation of international legal discourse applying the transformational model of translation analysis and the model involving tertium comparacionis - the language-mediator as a standard for comparing the original and target texts. The aim of the article is to highlight the peculiarities of the application of the transformation model and the model of the reference set of features for translation analysis of international legal discourse. The article used a complex research methodology involving methods of translation analysis, componential analysis, structural transformational analysis, as well as the elements of comparative method and text-interpretive analysis. The study reached the following conclusions. First, the transformation of nuclear structures of the English original text into surface text structures of the Ukrainian translation, carried out in accordance with the three-stage model of transformational analysis, shows the need to reproduce in Ukrainian translation conditional relations implied in the form of explanatory or attributive meanings. It is found that none of the obtained transformational structures completely coincides with the official translation into Ukrainian. In addition, the Ukrainian translation omits the modal verb of obligation shall, which is present in the English original text, so that the fragment of the translation loses the connotations of imperative modality, which does not correspond to the style of the translated text. The application of the translation analysis model related to the identification of the reference set of features for the original and target texts showed the presence of both common and different features from the reference set. The difference in the text of the translation is revealed by such allomorphic features as (a) "object of action in the singular", in contrast to the original fragment with the object of action in the plural, (b) change from modality of possibility to epistemic modality with verb in the present tense, as well as (c) implicated condition relations in the target Ukrainian text, in contrast to the explicit expression of the condition of the text of the original.