Actively transcribed rDNA and distal junction (DJ) sequence are involved in association of NORs with nucleoli.

Although they are organelles without a limiting membrane, nucleoli have an exclusive structure, built upon the rDNA-rich acrocentric short arms of five human chromosomes (nucleolar organizer regions or NORs). This has raised the question: what are the structural features of a chromosome required for its inclusion in a nucleolus? Previous work has suggested that sequences adjacent to the tandemly repeated rDNA repeat units (DJ, distal junction sequence) may be involved, and we have extended such studies by addressing several issues related to the requirements for the association of NORs with nucleoli. We exploited both a set of somatic cell hybrids containing individual human acrocentric chromosomes and a set of Human Artificial Chromosomes (HACs) carrying different parts of a NOR, including an rDNA unit or DJ or PJ (proximal junction) sequence. Association of NORs with nucleoli was increased when constituent rDNA was transcribed and may be also affected by the status of heterochromatin blocks formed next to the rDNA arrays. Furthermore, our data suggest that a relatively small size DJ region, highly conserved in evolution, is also involved, along with the rDNA repeats, in the localization of p-arms of acrocentric chromosomes in nucleoli. Thus, we infer a cooperative action of rDNA sequence-stimulated by its activity-and sequences distal to rDNA contributing to incorporation into nucleoli. Analysis of NOR sequences also identified LncRNA_038958 in the DJ, a candidate transcript with the region of the suggested promoter that is located close to the DJ/rDNA boundary and contains CTCF binding sites. This LncRNA may affect RNA Polymerase I and/or nucleolar activity. Our findings provide the basis for future studies to determine which RNAs and proteins interact critically with NOR sequences to organize the higher-order structure of nucleoli and their function in normal cells and pathological states.

Sharp focusing of partially coherent Bessel-correlated beams by a graded-index lens.

The nonparaxial focusing of partially coherent Bessel-correlated beams carrying vortices by a graded-index lens is investigated using the decomposition of the incident field into coherent modes and the quantum mechanical operator method. The influence of the coherence state and the incident beam aperture on tight focusing is analyzed. Our results show that a partially coherent Bessel-correlated beam can be focused into a spot of smaller size than coherent light.

A novel assay to screen siRNA libraries identifies protein kinases required for chromosome transmission.

One of the hallmarks of cancer is chromosome instability (CIN), which leads to aneuploidy, translocations, and other chromosome aberrations. However, in the vast majority of human tumors the molecular basis of CIN remains unknown, partly because not all genes controlling chromosome transmission have yet been identified. To address this question, we developed an experimental high-throughput imaging (HTI) siRNA assay that allows the identification of novel CIN genes. Our method uses a human artificial chromosome (HAC) expressing the GFP transgene. When this assay was applied to screen an siRNA library of protein kinases, we identified PINK1, TRIO, IRAK1, PNCK, and TAOK1 as potential novel genes whose knockdown induces various mitotic abnormalities and results in chromosome loss. The HAC-based assay can be applied for screening different siRNA libraries (cell cycle regulation, DNA damage response, epigenetics, and transcription factors) to identify additional genes involved in CIN. Identification of the complete spectrum of CIN genes will reveal new insights into mechanisms of chromosome segregation and may expedite the development of novel therapeutic strategies to target the CIN phenotype in cancer cells.

Human artificial chromosome (HAC) for measuring chromosome instability (CIN) and identification of genes required for proper chromosome transmission.

Chromosomal instability (CIN) is one of the characteristics of cancer inherent for tumor initiation and progression, which is defined as a persistent, high rate of gain/loss of whole chromosomes. In the vast majority of human tumors the molecular basis of CIN remains unknown. The development of a conceptually simple colony color sectoring assay that measures yeast artificial chromosome (YAC) loss provided a powerful genetic tool to assess the rate of chromosome mis-segregation and also identified 937 yeast genes involved in this process. Similarly, a human artificial chromosome (HAC)-based assay has been recently developed and applied to quantify chromosome mis-segregation events in human cells. This assay allowed identification of novel human CIN genes in the library of protein kinases. Among them are PINK1, TRIO, IRAK1, PNCK, and TAOK1. The HAC-based assay may be applied to screen siRNA, shRNA and CRISPR-based libraries to identify the complete spectrum of CIN genes. This will reveal new insights into mechanisms of chromosome segregation and may expedite the development of novel therapeutic strategies to target the CIN phenotype in cancer cells.

Small-Sized Interferometer with Fabry-Perot Resonators for Gravitational Wave Detection.

It is highly desirable to have a compact laser interferometer for detecting gravitational waves. Here, a small-sized tabletop laser interferometer with Fabry-Perot resonators consisting of two spatially distributed "mirrors" for detecting gravitational waves is proposed. It is shown that the spectral resolution of 10-23 cm-1 can be achieved at a distance between mirrors of only 1-3 m. The influence of light absorption in crystals on the limiting resolution of such resonators is also studied. A higher sensitivity of the interferometer to shorter-wave laser radiation is shown. A method for detecting gravitational waves is proposed based on the measurement of the correlation function of the radiation intensities of non-zero-order resonant modes from the two arms of the Mach-Zehnder interferometer.

Large positive and negative Goos-Hänchen shifts near the surface plasmon resonance in subwavelength grating.

Diffraction of light of a visible spectral range by subwavelength metal gratings has been investigated experimentally and theoretically using rigorous electromagnetic calculations. It has been demonstrated that an effect of surface plasmon resonance (SPR), at which total absorption of light by metal grating can be observed, occurs under certain conditions. Large positive and negative Goos-Hänchen (GH) shifts occur near the SPR. It has been shown that the reflected beam is split into two parts, the relative powers of which depend on the incident beam width and the grating depth. The dependence of the GH shifts on the grating period and grating depth has been investigated for different incident beam widths. The high sensitivity of the GH shift on the incident angle of a light beam near the SPR has been demonstrated.

Diffraction of partially-coherent light beams by microlens arrays.

The synthesis method including wave-optics and ray-tracing for the acceleration of the simulation of micro-optical systems has been developed. The effects of the spatial coherence and randomization of microlens array (MLA) parameters have been considered. The method based on coherent states representation for the calculation of the optical efficiency of microlens arrays taking into account the light source polarization has been developed. Numerical simulations of the intensity distributions and spreading angle of a diffracted beam have been carried out.

Primary cultures of human colon cancer as a model to study cancer stem cells.

The principal cause of death in cancer involves tumor progression and metastasis. Since only a small proportion of the primary tumor cells, cancer stem cells (CSCs), which are the most aggressive, have the capacity to metastasize and display properties of stem cells, it is imperative to characterize the gene expression of diagnostic markers and to evaluate the drug sensitivity in the CSCs themselves. Here, we have examined the key genes that are involved in the progression of colorectal cancer and are expressed in cancer stem cells. Primary cultures of colorectal cancer cells from a patient's tumors were studied using the flow cytometry and cytological methods. We have evaluated the clinical and stem cell marker expression in these cells, their resistance to 5-fluorouracil and irinotecan, and the ability of cells to form tumors in mice. The data shows the role of stem cell marker Oct4 in the resistance of primary colorectal cancer tumor cells to 5-fluorouracil.

Vector Laguerre-Gauss beams with polarization-orbital angular momentum entanglement in a graded-index medium.

It is shown that the vector-vortex Laguerre-Gauss modes with polarization-orbital angular momentum (OAM) entanglement are the vector solutions of the Maxwell equations in a graded-index medium. Focusing of linearly and circularly polarized vortex light beams with nonzero azimuthal and radial indices in a cylindrical graded-index waveguide is investigated. The wave shape variation with distance taking into account the spin-orbit and nonparaxial effects is analyzed. The effect of long-term periodic revival of wave packets due to mode interference in a graded-index cylindrical optical waveguide is demonstrated. High efficiency transfer of a strongly focused spot through an optical waveguide over large distances takes place with a period of revival.

Subwavelength diffractive color beam combiner.

A high-efficiency subwavelength diffractive beam combiner operating in a visible spectral range is designed, fabricated, and demonstrated. Such a device combines red, green, and blue color beams into one output light beam. Diffraction efficiencies of different types of gratings are calculated for various materials, incidence angles, and polarizations of light. It is shown that the plasmon resonance via a grating coupling occurs at the determined conditions. Subwavelength gratings with a period of 400 nm are fabricated and tested using laser and laser diode sources.

Splitting of levels in a cylindrical dielectric waveguide.

A splitting of modes in a cylindrical graded-index optical fiber is demonstrated by solving the full Maxwell equations using the perturbation analysis. It is shown that the degeneracy of vortex Laguerre-Gauss modes with distinct orbital angular momentum and polarization (spin) but the same total angular momentum is lifted due to the spin-orbit (vector) and tensor forces. Numerical estimations of group delays of modes in optical fiber and frequency splitting in Fabry-Perot and ring resonators are presented.

Nonparaxial Propagation of Bessel Correlated Vortex Beams in Free Space.

The nonparaxial propagation of partially coherent beams carrying vortices in free space is investigated using the method of decomposition of the incident field into coherent diffraction-free modes. Modified Bessel correlated vortex beams with the wavefront curvature are introduced. Analytical expressions are presented to describe the intensity distribution and the degree of coherence at different distances. The evolution of the intensity distribution during beam propagation for various source parameters is analyzed. The effects of nonparaxiality in the propagation of tightly focused coherent vortex beams are analyzed.

Transient expression of inactive RB in mesenchymal stem cells impairs their adipogenic potential and is associated with hypermethylation of the PPARγ2 promoter.

The retinoblastoma gene product (pRb) is a chromatin-associated protein that can either suppress or promote activity of key regulators of tissue-specific differentiation. We found that twelve weeks after transfection of the exogenous active (ΔB/X and Δр34) or inactive (ΔS/N) forms of RB into the 10T1/2 mesenchymal stem cells and clonal selection not a single cell line did contain exogenous RB, despite being G-418 resistant. However, the consequences of the transient production of exogenous RB had different effects on the cell fate. The ΔB/X and Δр34 cells transfected with active form of RB showed elevated levels of inducible adipocyte differentiation (AD). On the contrary, the ΔS/N cells transfected with inactive RB mutant were insensitive to induction of AD associated with abolishing of expression of the PPARγ2. Additionally, the PPARγ2 promoter in undifferentiated ΔS/N cells was hypermethylated, but all except -60 position CpG became mostly demethylated after cells exposure to AD. We conclude that while transient expression of inactive exogenous RB induces long term epigenetic alterations that prevent adipogenesis, production of active exogenous RBs results in an AD-promoting epigenetic state. These results indicate that pRb is involved in the establishment of hereditary epigenetic memory at least by creating a methylation pattern of PPARγ2.

The 1:1 host-guest complexation between cucurbit[7]uril and styryl dye.

The photophysical properties of aqueous solution of styryl dye, 4-[(E)-2-(3,4-dimethoxyphenyl)ethenyl]-1-ethylpyridinium perchlorate (dye 1), in the presence of cucurbit[7]uril (CB[7]) was studied by means of fluorescence spectroscopy methods. The production of 1:1 host-guest complexes in the range of CB[7] concentrations up to 16 µM with K = 1.0 × 10(6) M(-1) has been observed, which corresponds to appearance of the isosbestic point at 396 nm in the absorption spectra and a 5-fold increase in fluorescence intensity. The decay of fluorescence was found to fit to double-exponential functions in all cases; the calculated average fluorescence lifetime increases from 145 to 352 ps upon the addition of CB[7]. Rotational relaxation times of dye 1 solutions 119 ± 14 ps without CB[7] and 277 ± 35 ps in the presence of CB[7] have been determined by anisotropy fluorescence method. The comparison of the results of quantum-chemical calculations and experimental data confirms that in the host cavity dye 1 rotates as a whole with CB[7].

Assembly of Multiple Full-Size Genes or Genomic DNA Fragments on Human Artificial Chromosomes Using the Iterative Integration System.

Human artificial chromosomes (HACs) are gene delivery vectors that have been used for decades for gene functional studies. HACs have several advantages over viral-based gene transfer systems, including stable episomal maintenance in a single copy in the cell and the ability to carry up to megabase-sized genomic DNA segments. We have previously developed the alphoidtetO -HAC, which has a single gene acceptor loxP site that allows insertion of an individual gene of interest using Chinese hamster ovary (CHO) hybrid cells. The HAC, along with a DNA segment of interest, can then be transferred from donor CHO cells to various recipient cells of interest via microcell-mediated chromosome transfer (MMCT). Here, we detail a protocol for loading multiple genomic DNA segments or genes into the alphoidtetO -HAC vector using an iterative integration system (IIS) that utilizes recombinases Cre, ΦC31, and ΦBT. This IIS-alphoidtetO -HAC can be used for either serially assembling genomic loci or fragments of a large gene, or for inserting multiple genes into the same artificial chromosome. The insertions are executed iteratively, whereby each round results in the insertion of a new DNA segment of interest. This is accompanied by changes of expression of marker fluorescent proteins, which simplifies screening of correct clones, and changes of selection and counterselection markers, which constitutes an error-proofing mechanism that removes mis-incorporated DNA segments. In addition, the IIS-alphoidtetO -HAC carrying the genes can be eliminated from the cells, offering the possibility to compare the phenotypes of human cells with and without functional copies of the genes of interest. The resulting HAC molecules may be used to investigate biomedically relevant pathways or the regulation of multiple genes, and to potentially engineer synthetic chromosomes with a specific set of genes of interest. The IIS-alphoidtetO -HAC system is expected to be beneficial in creating multiple-gene humanized models with the purpose of understanding complex multi-gene genetic disorders. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Integration of the first DNA segment of interest into the IIS-alphoidteto -HAC Basic Protocol 2: Integration of a second DNA segment of interest into the IIS-alphoidteto -HAC Basic Protocol 3: Integration of a third DNA segment of interest into the IIS-alphoidteto -HAC Support Protocol: Fluorescence in situ hybridization analysis for the circular IIS-alphoidtetO -HAC.

Terpyridine platinum compounds induce telomere dysfunction and chromosome instability in cancer cells.

Telomerase/telomere-targeting therapy is a potentially promising approach for cancer treatment because even transient telomere dysfunction can induce chromosomal instability (CIN) and may be a barrier to tumor growth. We recently developed a dual-HAC (Human Artificial Chromosome) assay that enables identification and ranking of compounds that induce CIN as a result of telomere dysfunction. This assay is based on the use of two isogenic HT1080 cell lines, one carrying a linear HAC (containing telomeres) and the other carrying a circular HAC (lacking telomeres). Disruption of telomeres in response to drug treatment results in specific destabilization of the linear HAC. Results: In this study, we used the dual-HAC assay for the analysis of the platinum-derived G4 ligand Pt-tpy and five of its derivatives: Pt-cpym, Pt-vpym, Pt-ttpy, Pt(PA)-tpy, and Pt-BisQ. Our analysis revealed four compounds, Pt-tpy, Pt-ttpy, Pt-vpym and Pt-cpym, that induce a specific loss of a linear but not a circular HAC. Increased CIN after treatment by these compounds correlates with the induction of double-stranded breaks (DSBs) predominantly localized at telomeres and reflecting telomere-associated DNA damage. Analysis of the mitotic phenotypes induced by these drugs revealed an elevated rate of chromatin bridges (CBs) in late mitosis and cytokinesis. These terpyridine platinum-derived G4 ligands are promising compounds for cancer treatment.

The genomic structure of a human chromosome 22 nucleolar organizer region determined by TAR cloning.

The rDNA clusters and flanking sequences on human chromosomes 13, 14, 15, 21 and 22 represent large gaps in the current genomic assembly. The organization and the degree of divergence of the human rDNA units within an individual nucleolar organizer region (NOR) are only partially known. To address this lacuna, we previously applied transformation-associated recombination (TAR) cloning to isolate individual rDNA units from chromosome 21. That approach revealed an unexpectedly high level of heterogeneity in human rDNA, raising the possibility of corresponding variations in ribosome dynamics. We have now applied the same strategy to analyze an entire rDNA array end-to-end from a copy of chromosome 22. Sequencing of TAR isolates provided the entire NOR sequence, including proximal and distal junctions that may be involved in nucleolar function. Comparison of the newly sequenced rDNAs to reference sequence for chromosomes 22 and 21 revealed variants that are shared in human rDNA in individuals from different ethnic groups, many of them at high frequency. Analysis infers comparable intra- and inter-individual divergence of rDNA units on the same and different chromosomes, supporting the concerted evolution of rDNA units. The results provide a route to investigate further the role of rDNA variation in nucleolar formation and in the empirical associations of nucleoli with pathology.

p130 And pRb in the Maintenance of Transient Quiescence of Mesenchymal Stem Cells.

An effective regulation of quiescence plays a key role in the differentiation, plasticity, and prevention of stem cells from becoming malignant. The state of quiescence is being controlled by the pRb family proteins which show overlapping functions in cell cycle regulation; however, their roles in controlling the proliferation of mesenchymal stem cells (MSCs) remain to be understood. This study investigated the regulation of transient quiescence using growth curves, proliferation assay, the cytometric evaluation of cell cycle, Western blotting, and the electromobility gel shift assay (EMSA) on synchronized MSCs of the C3H10Т1/2 and control cells with different statuses of pRb proteins. It has been found that functional steady-state level of p130 but not pRb plays a critical role for entering, exiting, and maintenance of transient quiescence in multipotent mesenchymal stem cells.

Preferential solvation of spherical ions in binary DMSO/benzene mixtures.

We consider a new qualitative approach for treating theoretically the solvation of single-atomic ionic solutes in binary mixtures of polar and nonpolar aprotic solvents. It is based on the implicit continuum electrostatic model of the solvent mixture involving distance-dependent dielectric permittivity epsilon(R) (where R is the distance from the ion) and local concentrations C(1)(R) and C(2)(R) of the solvent ingredients. For a given R, the condition for local thermodynamic equilibrium provides the transcendental equation for explicitly establishing the permittivity and concentration profiles. Computations performed with real Cl(-) and model Cl(+) ions as solutes in benzene/DMSO mixtures are compared with the molecular dynamics simulations of the same systems. A significant discrepancy of molecular and continuum results is revealed for the concentration profiles in the close vicinity of the ion boundary, although the general trends are similar. The continuum methodology cannot account for the formation of rigid solvent structures around ions, which is most significant for the case of Cl(+). Such defect, however, proves to become of less importance in calculations of the solvation free energy, which are quite satisfactory for Cl(-) ion. Free energy calculations for Cl(+) are less successful in the range of low DMSO concentration.

Advanced dielectric continuum model of preferential solvation.

A continuum model for solvation effects in binary solvent mixtures is formulated in terms of the density functional theory. The presence of two variables, namely, the dimensionless solvent composition y and the dimensionless total solvent density z, is an essential feature of binary systems. Their coupling, hidden in the structure of the local dielectric permittivity function, is postulated at the phenomenological level. Local equilibrium conditions are derived by a variation in the free energy functional expressed in terms of the composition and density variables. They appear as a pair of coupled equations defining y and z as spatial distributions. We consider the simplest spherically symmetric case of the Born-type ion immersed in the benzene/dimethylsulfoxide (DMSO) solvent mixture. The profiles of y(R) and z(R) along the radius R, which measures the distance from the ion center, are found in molecular dynamics (MD) simulations. It is shown that for a given solute ion z(R) does not depend significantly on the composition variable y. A simplified solution is then obtained by inserting z(R), found in the MD simulation for the pure DMSO, in the single equation which defines y(R). In this way composition dependences of the main solvation effects are investigated. The local density augmentation appears as a peak of z(R) at the ion boundary. It is responsible for the fine solvation effects missing when the ordinary solvation theories, in which z=1, are applied. These phenomena, studied for negative ions, reproduce consistently the simulation results. For positive ions the simulation shows that z>>1 (z=5-6 at the maximum of the z peak), which means that an extremely dense solvation shell is formed. In such a situation the continuum description fails to be valid within a consistent parametrization.