Elena I. Koshel

Coordination to Imidazole Ring Switches on Phosphorescence of Platinum Cyclometalated Complexes: The Route to Selective Labeling of Peptides and Proteins via Histidine Residues

In this study, we have shown that substitution of chloride ligand for imidazole (Im) ring in the cyclometalated platinum complex Pt(phpy)(PPh3)Cl (1; phpy, 2-phenylpyridine; PPh3, triphenylphosphine), which is nonemissive in solution, switches on phosphorescence of the resulting compound. Crystallographic and nuclear magnetic resonance (NMR) spectroscopic studies of the substitution product showed that the luminescence ignition is a result of Im coordination to give the [Pt(phpy)(Im)(PPh3)]Cl complex. The other imidazole-containing biomolecules, such as histidine and histidine-containing peptides and proteins, also trigger luminescence of the substitution products. The complex 1 proved to be highly selective toward the imidazole ring coordination that allows site-specific labeling of peptides and proteins with 1 using the route, which is orthogonal to the common bioconjugation schemes via lysine, aspartic and glutamic acids, or cysteine and does not require any preliminary modification of a biomolecule. The utility of this approach was demonstrated on (i) site-specific modification of the ubiquitin, a small protein that contains only one His residue in its sequence, and (ii) preparation of nonaggregated HSA-based Pt phosphorescent probe. The latter particles easily internalize into the live HeLa cells and display a high potential for live-cell phosphorescence lifetime imaging (PLIM) as well as for advanced correlation PLIM and FLIM experiments.

Chicken rRNA Gene Cluster Structure

Ribosomal RNA (rRNA) genes, whose activity results in nucleolus formation, constitute an extremely important part of genome. Despite the extensive exploration into avian genomes, no complete description of avian rRNA gene primary structure has been offered so far. We publish a complete chicken rRNA gene cluster sequence here, including 5’ETS (1836 bp), 18S rRNA gene (1823 bp), ITS1 (2530 bp), 5.8S rRNA gene (157 bp), ITS2 (733 bp), 28S rRNA gene (4441 bp) and 3’ETS (343 bp). The rRNA gene cluster sequence of 11863 bp was assembled from raw reads and deposited to GenBank under KT445934 accession number. The assembly was validated through in situ fluorescent hybridization analysis on chicken metaphase chromosomes using computed and synthesized specific probes, as well as through the reference assembly against de novo assembled rRNA gene cluster sequence using sequenced fragments of BAC-clone containing chicken NOR (nucleolus organizer region). The results have confirmed the chicken rRNA gene cluster validity.

Chicken Microchromosomes in the Lampbrush Phase: A Cytogenetic Description

Lampbrush chromosomes are giant, transcriptionally active, meiotic chromosomes found in oocytes of all vertebrates with the exception of mammals. Lampbrush chromosomes offer a convenient tool for cytogenetic mapping and, in particular, have been instrumental in mapping genes and linkage groups on chicken (GGA) chromosomes. Whereas cytogenetic maps of macrochromosome GGA1-10 and microchromosome GGA11-16 lampbrush bivalents have been established, identification and description of smaller microchromosome bivalents are still missing. In this work, we used specific FISH probes for the identification of 12 chicken lampbrush chromosomes formed by GGA17-28. Our observations on chromomere and lateral loop arrangement and chiasma position allowed us to construct the respective cytogenetic maps for these microchromosomes. For the 10 smallest chicken microchromosomes, GGA29-38, no individual molecular tags are available, yet they can be collectively marked using the PO41 repeat. The reported results contribute to building of working cytogenetic maps of the chicken karyotype.

Ribosomal RNA gene functioning in avian oogenesis.

Despite long-term exploration into ribosomal RNA gene functioning during the oogenesis of various organisms, many intriguing problems remain unsolved. In this review, we describe nucleolus organizer region (NOR) activity in avian oocytes. Whereas oocytes from an adult avian ovary never reveal the formation of the nucleolus in the germinal vesicle (GV), an ovary from juvenile birds possesses both nucleolus-containing and non-nucleolus-containing oocytes. The evolutionary diversity of oocyte NOR functioning and the potential non-rRNA-related functions of the nucleolus in oocytes are also discussed.

How to avoid protein aggregation to improve cellular uptake of albumin-based conjugates: towards the rational design of cell-penetrable phosphorescent probes

Albumins are very promising carrier family for various types of small functional molecules, including (pro)drugs, sensors, and imaging agents. In this study, we demonstrate that one of the most crucial features of albumin—its ability to internalize into cancer cells—is strongly affected by its aggregation state. We used 14 different luminescent complexes (polynuclear heterometallic Ag(I)-Au(I) and Cu(I)-Au(I) clusters and a series of mononuclear water-soluble and hydrophobic Pt(II) complexes) able to form either covalent conjugates or non-covalent adducts with human serum albumin (HSA) to investigate the impact of preparation conditions on aggregation behavior of HSA. We demonstrated that preparation of non-aggregated HSA-based conjugates/adducts requires compliance with rather strict reaction conditions (aqueous solution with as low as possible organic co-solvent content and complex/HSA molar ratio not exceeding some critical value) while bypassing these rules (e.g., using the reaction mixtures enriched by organic solvent, high complex/HSA molar ratio or bulky hydrophobic molecules) may lead to HSA aggregation or even instability of the conjugates in solution. Further, we showed that non-aggregated HSA conjugates with water-soluble Pt complex easily internalize into cancer HeLa cells, while fully aggregated non-covalent HSA adducts with heterometallic Cu-Au clusters do not. Ultimately, in this study, we put forward two major points. First, we formulate general rules providing simple preparation route for non-aggregated cell-penetrable HSA-based conjugates, particularly, phosphorescent probes. Second, we demonstrate that the aggregation state of albumin is a very important parameter, which should be controlled while preparing its conjugates.

New high copy tandem repeat in the content of the chicken W chromosome

The content of repetitive DNA in avian genomes is considerably less than in other investigated vertebrates. The first descriptions of tandem repeats were based on the results of routine biochemical and molecular biological experiments. Both satellite DNA and interspersed repetitive elements were annotated using library-based approach and de novo repeat identification in assembled genome. The development of deep-sequencing methods provides datasets of high quality without preassembly allowing one to annotate repetitive elements from unassembled part of genomes. In this work, we search the chicken assembly and annotate high copy number tandem repeats from unassembled short raw reads. Tandem repeat (GGAAA)n has been identified and found to be the second after telomeric repeat (TTAGGG)n most abundant in the chicken genome. Furthermore, (GGAAA)n repeat forms expanded arrays on the both arms of the chicken W chromosome. Our results highlight the complexity of repetitive sequences and update data about organization of sex W chromosome in chicken.

Nanostructured Layer-by-Layer Polyelectrolyte Containers to Switch Biofilm Fluorescence

The development of stimuli-responsive nanocontainers is an issue of utmost importance for many applications such as targeted drug delivery, regulation of the cell and tissue behavior, making bacteria have useful functions and here converting light. The present work shows a new contribution to the design of polyelectrolyte (PE) containers based on surface modified mesoporous titania particles with deposited Ag nanoparticles to achieve chemical light upconversion via biofilms. The PE shell allows slowing down the kinetics of a release of loaded l-arabinose and switching the bacteria luminescence in a certain time. The hybrid TiO2/Ag/PE containers activated at 980 nm (IR) illumination demonstrate 10 times faster release of l-arabinose as opposed to non-activated containers. Fast IR-released l-arabinose switch bacteria fluorescence which we monitor at 510 nm. The approach described herein can be used in many applications where the target and delayed switching and light upconversion are required.

Functional features of the nucleolar organizer in developing oocytes of juvenile birds

The genes of rRNA in the nucleolar organizer region (NOR) are inactivated in the oocytes of adult birds despite the functioning of lampbrush chromosomes. The nucleolus is not formed during all stages of the oocyte development. On the other hand, two morphological forms of oocytes differing by the presence of nucleolus in the germinal vesicle are described in the ovaries of juvenile birds. The activation and function of the ribosomal genes in avian oogenesis is still vague. In this work, the NOR activation in chicken (Gallus gallus domesticus) oocytes is confirmed with the help of fluorescence immunohistochemistry (antibodies against nucleophosmin, fibrillarin, and UBF1) and in situ nucleic acid hybridization (FISH with the probe to ITS1 in pre-rRNA). It is demonstrated that the nucleolus in the oocytes at the lampbrush stage in the chicken ovaries is fragmented after complete inactivation of the ribosome genes: the nucleolar fragments contain fibrillarin but do not contain pre-rRNA molecule. The utility of the ovary 3D reconstruction using serial histological sections for quantification of sex cell population heterogeneity in the ovaries of juvenile birds is demonstrated. The obtained results improve the current insight into the functional NOR state in the oocytes of juvenile female birds and contribute to the concept of diversity in the scenarios of gametogenesis.

Evolution of ribosomal internal transcribed spacers in Deuterostomia

Sequences of ribosomal internal transcribed spacers (ITSs) are of great importance to molecular phylogenetics and DNA barcoding, but remain unstudied in some large taxa of Deuterostomia. We have analyzed complete ITS1 and ITS2 sequences in 62 species from 16 Deuterostomia classes, with ITS sequences in 24 species from 11 classes initially obtained using unannotated contigs and raw read sequences. A general tendency for both ITS length and GC-content increase from interior to superior Deuterostomia taxa, a uniform GC-content in both ITSs within the same species, thymine content decrease in sense DNA sequences of both ITSs are shown. A possible role of GC-based gene conversion in Deuterostomia ITS evolutionary changes is hypothesized. The first example of non-LTR retrotransposon insertion into ITS sequence in Deuterostomia is described in turtle Geochelone nigra. The roles of mobile genetic element insertions in the evolution of ITS sequences in some Sauropsida taxa are discussed as well.