improved-genome-editing-via-oviductal-nucleic-acids-delivery-based

Improved Genome Editing <em>via</em> Oviductal Nucleic Acids Delivery-based <em>In Vivo</em> Electroporation Technique for Knockout Mice Generation

Methods for creating knockout mice typically involve three main steps: (1) collecting embryos from donor females, (2) microinjecting genetic constructs ...

St. Petersburg State University

We analyzed the phylogenetic relationships of the genus Trisetacus using two genes [cytochrome c oxidase subunit I (COI) and D1-D2 region of 28S rDNA (D1-D2 28S)], a representive taxon sampling (nearly 40% of known diversity), and a large set of close and distant outgroups. Our analyses suggest the presence of a dichotomy between Trisetacus associated with Cupressaceae and Pinaceae. The following smaller molecular clades were found: Pin-1 (bud mites, twig sheath mites, bark gall mites, and endoparasitic mites from pinaceans), Pin-2 (needle sheath mites from pines), Pin-2a (putative Nearctic group of needle sheath mites), Pin-2b (putative Palearctic group of needle sheath mites), Cup-1 and 2 (bud, cone, seed mites and mites living under bark scales from cupressaceans). The monophyly of the recently proposed subgenus Brevithecus nested within clade Cup-2 was confirmed. Ancestral character reconstruction analyses recovered: (1) Pinaceae as the ancestral hosts of Nalepellidae and Trisetacus, (2) repetitive reductions of the spermathecal tube independently occurred in two lineages of Trisetacus from Cupressaceae, and (3) several mite habitats on host (galls, cones, twig sheaths, seeds, inside leaves, and under scales) are evolutionarily derived states, whereas living in buds or needle sheaths are ancestral states for Trisetacus clades Cup and Pin. Using confocal microscopy, we identified six basic types of the female internal genitalia of Trisetacus based on shapes of the spermatheca and spermathecal tube. These genitalic types are strongly correlated with lineages recovered by molecular phylogenetic analyses, suggesting that the female genital morphology is both evolutionarily conserved and is a factor influencing macroevolutionary patterns in this group of mites.

Molecular phylogenetic analyses reveal a deep dichotomy in the conifer-inhabiting genus Trisetacus (Eriophyoidea: Nalepellidae), with the two lineages differing in their female genital morphology and host associations.

Phytoptidae s.str. is a lineage of eriophyoid mites associated with angiosperms. Based on representative taxon sampling and four gene markers (COI, HSP70, 18S, and 28S), we inferred the molecular phylogeny of this group and performed comparative analyses of cuticle-lined female internal genitalia. Although basal relationships were unclear, several well supported clades were recovered. These clades were supported by geography, host associations, and female genital anatomy, but contradicted the current morphology-based systematics. The monophyly of each of five conventional supraspecific groupings (Fragariocoptes, Phytoptus, Phytoptinae, Sierraphytoptinae, and Sierraphytoptini) is rejected based on a series of statistical tests. Additionally, four morphological characters (the absence of tibial solenidion φ and opisthosomal seta c1, presence of telosomal pseudotagma, and 'morphotype') were found to be homoplasies that cannot be used to confidently delimit supraspecific lineages of phytoptids. However, our molecular topology was highly congruent with female genital characters. Eight molecular clades were unambiguously supported by the shapes and topography of the spermathecal apparatus and genital apodemes. This suggests that the female genital anatomy could be an important factor affecting cladogenesis in Phytoptidae, a conclusion contrasting with the general expectation that host characteristics should be a major macroevolutionary force influencing the evolution of host-specific symbionts. Indeed, despite the high host-specificity, there were no apparent cophylogenetic patterns. Furthermore, we show that gall-inducing ability evolved multiple times in phytoptids. Because gall formation creates nearly instantaneous niche partitioning and the potential loss or reduction of gene flow, we hypothesize that it could be an important evolutionary factor affecting speciation within different host-associated clades of phytoptid mites.

Molecular phylogeny of the phytoparasitic mite family Phytoptidae (Acariformes: Eriophyoidea) identified the female genitalic anatomy as a major macroevolutionary factor and revealed multiple origins of gall induction.

Plant photobodies are the membrane-less organelles (MLOs) that can be generated by protein-protein interactions between active form of phytochrome B (phyB) and phytochrome-interacting factors (PIFs). These organelles regulate plant photomorphogenesis. In this study, we developed two chimeric proteins with fluorescent proteins, phyB fused to EGFP and PIF6 fused to mCherry, and investigated their exogenous expression in mammalian cells by confocal fluorescence microscopy. Results showed that irradiation with diffused 630-nm light induced formation and subsequent increase in sizes of the MLOs. The assembly and disassembly of the photo-inducible MLOs in the mammalian cell cytoplasm obeyed the laws inherent in the concentration-dependent phase separation of biopolymers. The sizes of MLOs formed from phyB and PIF6 in mammalian cells corresponded to the sizes of the so-called "early" photobodies in plant cells. These results suggested that the first step for the formation of plant photobodies might be based on the light-dependent liquid-liquid phase separation of PIFs and other proteins that can specifically interact with the active form of phyB. The developed chimeric proteins in principle can be used to control the assembly and disassembly of photo-inducible MLOs, and thereby to regulate various intracellular processes in mammalian cells.

Photo-dependent membrane-less organelles formed from plant phyB and PIF6 proteins in mammalian cells.

In this work, we put forward a hypothesis about the decisive role of multivalent nonspecific interactions in the early stages of PML body formation. Our analysis of the PML isoform sequences showed that some of the PML isoforms, primarily PML-II, are prone to phase separation due to their polyampholytic properties and the disordered structure of their C-terminal domains. The similarity of the charge properties of the C-terminal domains of PML-II and PML-VI isoforms made it possible for the first time to detect migration of PML-VI from PML bodies to the periphery of the cell nucleus, similar to the migration of PML-II isoforms. We found a population of "small" (area less than 1 µm) spherical PML bodies with high dynamics of PML isoforms exchange with nucleoplasm and a low fraction of immobilized proteins, which indicates their liquid state properties. Such structures can act as "seeds" of functionally active PML bodies, providing the necessary concentration of PML isoforms for the formation of intermolecular disulfide bonds between PML monomers. FRAP analysis of larger bodies of toroidal topology showed the existence of an insoluble scaffold in their structure. The hypothesis about the role of nonspecific multiple weak interactions in the formation of PML bodies is further supported by the change in the composition of the scaffold proteins of PML bodies, but not their solidification, under conditions of induction of dimerization of PML isoforms under oxidative stress. Using the colocalization of ALT-associated PML bodies (APBs) with TRF1, we identified APBs and showed the difference in the dynamic properties of APBs and canonical PML bodies.

The Role of Non-Specific Interactions in Canonical and ALT-Associated PML-Bodies Formation and Dynamics.

In this work, we performed a comparative study of the formation of PML bodies by full-length PML isoforms and their C-terminal domains in the presence and absence of endogenous PML. Based on the analysis of the distribution of intrinsic disorder predisposition in the amino acid sequences of PML isoforms, regions starting from the amino acid residue 395 (i.e., sequences encoded by exons 4-6) were assigned as the C-terminal domains of these proteins. We demonstrate that each of the full-sized nuclear isoforms of PML is capable of forming nuclear liquid-droplet compartments in the absence of other PML isoforms. These droplets possess dynamic characteristics of the exchange with the nucleoplasm close to those observed in the wild-type cells. Only the C-terminal domains of the PML-II and PML-V isoforms are able to be included in the composition of the endogenous PML bodies, while being partially distributed in the nucleoplasm. The bodies formed by the C-terminal domain of the PML-II isoform are dynamic liquid droplet compartments, regardless of the presence or absence of endogenous PML. The C-terminal domain of PML-V forms dynamic liquid droplet compartments in the knockout cells (PML), but when the C-terminus of the PML-V isoform is inserted into the existing endogenous PML bodies, the molecules of this protein cease to exchange with the nucleoplasm. It was demonstrated that the K490R substitution, which disrupts the PML sumoylation, promotes diffuse distribution of the C-terminal domains of PML-II and PML-V isoforms in endogenous PML knockout HeLa cells, but not in the wild-type cells. These data indicate the ability of the C-terminal domains of the PML-II and PML-V isoforms to form dynamic liquid droplet-like compartments, regardless of the ordered N-terminal RBCC motifs of the PML. This indicates a significant role of the non-specific interactions between the mostly disordered C-terminal domains of PML isoforms for the initiation of liquid-liquid phase separation (LLPS) leading to the formation of PML bodies.

New Evidence of the Importance of Weak Interactions in the Formation of PML-Bodies.

Eriophyoid mites of the genus  Keifer are widespread parasites of conifers. A new oligophagous species, &nbsp;, was discovered severely damaging seeds of North American junipers (, , and ) in the western USA. It has two codon deletions in the mitochondrial gene  rarely detected in Eriophyoidea and includes distinct morphological dimorphism of females. A phylogenetic analysis based on amino acid alignment of translated  sequences using a large set of out-groups (a) determined that two North American congeners,  and  were the closest known relatives of &nbsp;, and (b) indicated that Old and New World seed-inhabiting  from junipers do not form a distinct clade, suggesting a possible independent transition to living in seeds of junipers in America and Eurasia by  spp. Our analysis produced a new topology consistent with a scenario assuming gradual reduction of prodorsal shield setation in Eriophyoidea and an ancient switch from gymnosperms to other hosts. Additionally, our analysis did not support monophyly of ; recovered a new host-specific, moderately supported clade comprising  and Nalepellinae ( + ) associated with Pinaceae; and questioned the monophyly of  associated with Cupressaceae.

Phylogenetic Position of a New  Mite Species (Nalepellidae) Destroying Seeds of North American Junipers and New Hypotheses on Basal Divergence of Eriophyoidea.

The genus  Fabricius, 1807 includes numerous taxa and forms with uncertain status and taxonomic position. Among such taxa are  Alphéraky, 1897 and  Staudinger, 1897, interpreted in the literature either as conspecific forms, as subspecies of different but morphologically somewhat similar  species or as distinct species-level taxa. Based on mitochondrial and nuclear DNA markers, we reconstructed a phylogeographic pattern of the taxa in question. We recover and include in our analysis DNA barcodes of the century-old type specimens, the lectotype of  deposited in the Natural History Museum (Museum für Naturkunde), Berlin, Germany (ZMHU) and the paralectotype of  and the lectotype of  deposited in the Zoological Institute, Russian Academy of Sciences, St. Petersburg, Russia (ZISP). Our analysis grouped all specimens within four (HP_I-HP_IV) deeply divergent but geographically poorly structured clades which did not support nonconspecifity of -. We also show that all studied females of the widely distributed haplogroup HP_II were infected with a single  strain belonging to the supergroup B, while the males of this haplogroup, as well as all other investigated specimens of both sexes, were not infected. Our data highlight the relevance of large-scale sampling dataset analysis and the need for testing for  infection to avoid erroneous phylogenetic reconstructions and species misidentification.

Phylogeography of Two Enigmatic Sulphur Butterflies,  Alphéraky, 1897 and  Staudinger, 1897 (Lepidoptera, Pieridae), with Relations to  Infection.

At the molecular level, aging is often accompanied by dysfunction of stress-induced membrane-less organelles (MLOs) and changes in their physical state (or material properties). In this work, we analyzed the proteins included in the proteome of stress granules (SGs) and P-bodies for their tendency to transform the physical state of these MLOs. Particular attention was paid to the proteins whose gene expression changes during replicative aging. It was shown that the proteome of the studied MLOs consists of intrinsically disordered proteins, 30-40% of which are potentially capable of liquid-liquid phase separation (LLPS). Proteins whose gene expression changes during the transition of human cells to a senescent state make up about 20% of the studied proteomes. There is a statistically significant increase in the number of positively charged proteins in both datasets studied compared to the complete proteomes of these organelles. An increase in the relative content of DNA-, but not RNA-binding proteins, was also found in the SG dataset with senescence-related processes. Among SGs proteins potentially involved in senescent processes, there is an increase in the abundance of potentially amyloidogenic proteins compared to the whole proteome. Proteins common to SGs and P-bodies, potentially involved in processes associated with senescence, form clusters of interacting proteins. The largest cluster is represented by RNA-binding proteins involved in RNA processing and translation regulation. These data indicate that SG proteins, but not proteins of P-bodies, are more likely to transform the physical state of MLOs. Furthermore, these MLOs can participate in processes associated with aging in a coordinated manner.

Stress-granules, P-bodies, and cell aging: A bioinformatics study.

In the last decade, much attention was given to the study of physiological amyloid fibrils. These structures include A-bodies, which are the nucleolar fibrillar formations that appear in the response to acidosis and heat shock, and disassemble after the end of stress. One of the proteins involved in the biogenesis of A-bodies, regardless of the type of stress, is Von-Hippel Lindau protein (VHL). Known also as a tumor suppressor, VHL is capable to form amyloid fibrils both in vitro and in vivo in response to the environment acidification. As with most amyloidogenic proteins fusion with various tags is used to increase the solubility of VHL. Here, we first performed AFM-study of fibrils formed by VHL protein and by VHL fused with GST-tag (GST-VHL) at acidic conditions. It was shown that formed by full-length VHL fibrils are short heterogenic structures with persistent length of 2400 nm and average contour length of 409 nm. GST-tag catalyzes VHL amyloid fibril formation, superimpose chirality, increases length and level of hierarchy, but decreases rigidity of amyloid fibrils. The obtained data indicate that tagging can significantly affect the fibrillogenesis of the target protein.

Von-Hippel Lindau protein amyloid formation. The role of GST-tag.

The explosive growth in the number of works addressing the phase separation of intrinsically disordered proteins has driven both the development of new approaches and the optimization of existing methods for biomolecular condensate visualization. In this work, we studied the potential use of the fluorescent dye ANS as a sensor for liquid-liquid phase separation (LLPS), focusing on visualizing condensates formed by the stress-granules scaffold protein G3BP1. Using fluorescence lifetime imaging microscopy (FLIM), we demonstrated that ANS can accumulate in RNA-induced G3BP1 condensates in aqueous solutions, but not in G3BP1 condensates formed under macromolecular crowding conditions in highly concentrated PEG solutions. We showed that the experimentally determined limiting fluorescence anisotropy (r'), which characterizes the amplitude of high-frequency intramolecular mobility of ANS in aqueous solutions containing RNA-induced G3BP1 condensates, is half the value observed for ANS in aqueous G3BP1 solutions. Our results demonstrate the feasibility of using time-resolved fluorescence spectroscopy and microscopy of ANS for detecting LLPS of intrinsically disordered proteins in aqueous solutions.

Time-resolved fluorescence of ANS dye as a sensor of proteins LLPS.

Natural aging and age-related diseases involve the acceleration of replicative aging, or senescence. Multiple proteins are known to participate in these processes, including the promyelocytic leukemia (PML) protein, which serves as a core component of nuclear-membrane-less organelles known as PML nuclear bodies (PML-NBs). In this work, morphological changes in PML-NBs and alterations in PML protein localization at the transition of primary fibroblasts to a replicative senescent state were studied by immunofluorescence. The fibroblasts were obtained from both healthy donors and donors with premature aging syndromes (ataxia-telangiectasia and Cockayne syndrome). Our data showed an increase in both the size and the number of PML-NBs, along with nuclear enlargement in senescent cells, suggesting these changes could serve as potential cellular aging markers. Bioinformatic analysis demonstrated that 30% of the proteins in the PML interactome and ~45% of the proteins in the PML-NB predicted proteome are directly associated with senescence and aging processes. These proteins are hypothesized to participate in post-translational modifications and protein sequestration within PML-NBs, thereby influencing transcription factor regulation, DNA damage response, and negative regulation of apoptosis. The findings confirm the significant role of PML-NBs in cellular aging processes and open new avenues for investigating senescence mechanisms and age-associated diseases.

PML Nuclear Bodies and Cellular Senescence: A Comparative Study of Healthy and Premature Aging Syndrome Donors' Cells.

Based on molecular phylogenetic analysis of all relevant taxa, we propose to consider the species previously classified as members of Pseudophilotes, Palaeophilotes, Rubrapterus, and Inderskia as belonging to a single genus, the valid name of which is Palaeophilotes. This genus can be divided into two subgenera: Rubrapterus with species P. bavius and P. fatma, and Palaeophilotes sensu stricto. The latter subgenus includes four lineages and nine species: (1) the P. abencerragus lineage (single species P. abencerragus), (2) P. barbagiae lineage (single species P. barbagiae), (3) P. panope lineage (P. panope and P. triphysina), and (4) P. baton lineage (P. panoptes, P. baton, P. vicrama, P. jacuticus and P. sinaicus). The name Borisinia Korb, 2013, syn. nov. is shown to be an objective synonym of Palaeophilotes Forster, 1938. The previously proposed synonymy of P. svetlana and P. marina with P. panope is supported by the identity of their DNA-barcodes. Palaeophilotes panope is reported for the Kazakhstan part of the Altai mountains for the first time. Palaeophilotes jacuticus is confirmed for the Lake Baikal region in Siberia.

Taxonomy of the West Palaearctic butterfly genus Palaeophilotes Forster, 1938 (Lepidoptera: Lycaenidae) based on combined analysis of COI barcodes and multilocus nuclear markers.

Article	Year
Molecular phylogenetic analyses reveal a deep dichotomy in the conifer-inhabiting genus Trisetacus (Eriophyoidea: Nalepellidae), with the two lineages differing in their female genital morph... Experimental & applied acarology\| PubMed ID: 32514877	2020
Molecular phylogeny of the phytoparasitic mite family Phytoptidae (Acariformes: Eriophyoidea) identified the female genitalic anatomy as a major macroevolutionary factor and revealed multipl... Experimental & applied acarology\| PubMed ID: 33201392	2021
Photo-dependent membrane-less organelles formed from plant phyB and PIF6 proteins in mammalian cells. International journal of biological macromolecules\| PubMed ID: 33582218	2021
The Role of Non-Specific Interactions in Canonical and ALT-Associated PML-Bodies Formation and Dynamics. International journal of molecular sciences\| PubMed ID: 34072343	2021
New Evidence of the Importance of Weak Interactions in the Formation of PML-Bodies. International journal of molecular sciences\| PubMed ID: 35163537	2022
Phylogenetic Position of a New Mite Species (Nalepellidae) Destroying Seeds of North American Junipers and New Hypotheses on Basal Divergence of Eriophyoidea. Insects\| PubMed ID: 35206774	2022
Phylogeography of Two Enigmatic Sulphur Butterflies, Alphéraky, 1897 and Staudinger, 1897 (Lepidoptera, Pieridae), with Relations to Infection. Insects\| PubMed ID: 38132616	2023
Stress-granules, P-bodies, and cell aging: A bioinformatics study. Biochemical and biophysical research communications\| PubMed ID: 38147698	2024
Von-Hippel Lindau protein amyloid formation. The role of GST-tag. Biochemical and biophysical research communications\| PubMed ID: 38685186	2024
Time-resolved fluorescence of ANS dye as a sensor of proteins LLPS. Biochemical and biophysical research communications\| PubMed ID: 39673974	2025
PML Nuclear Bodies and Cellular Senescence: A Comparative Study of Healthy and Premature Aging Syndrome Donors' Cells. Cells\| PubMed ID: 39768166	2024
Taxonomy of the West Palaearctic butterfly genus Palaeophilotes Forster, 1938 (Lepidoptera: Lycaenidae) based on combined analysis of COI barcodes and multilocus nuclear markers. Zootaxa\| PubMed ID: 40174054	2025

Anna E. Romanovich

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