Characterization of CoCas9 nuclease from Capnocytophaga ochracea.

Cas9 nucleases are widely used for genome editing and engineering. Cas9 enzymes encoded by CRISPR-Cas defence systems of various prokaryotic organisms possess different properties such as target site preferences, size, and DNA cleavage efficiency. Here, we biochemically characterized CoCas9 from Capnocytophaga ochracea, a bacterium that inhabits the oral cavity of humans and contributes to plaque formation on teeth. CoCas9 recognizes a novel 5'-NRRWC-3' PAM and efficiently cleaves DNA in vitro. Functional characterization of CoCas9 opens ways for genetic engineering of C. ochracea using its endogenous CRISPR-Cas system. The novel PAM requirement makes CoCas9 potentially useful in genome editing applications.

[Type II CRISPR-Cas System Nucleases: A Pipeline for Prediction and In Vitro Characterization].

The use of CRISPR-Cas bacterial adaptive immunity system components for targeted DNA changes has opened broad prospects for programmable genome editing of higher organisms. The most widely used gene editors are based on the Cas9 effectors of the type II CRISPR-Cas systems. In complex with guide RNAs, Cas9 proteins are able to directionally introduce double-stranded breaks into DNA regions that are complementary to guide RNA sequences. Despite the wide range of characterized Cas9s, the search for new Cas9 variants remains an important task, since the available Cas9 editors have several limitations. This paper presents a workflow for the search for and subsequent characterization of new Cas9 nucleases developed in our laboratory. Detailed protocols describing the bioinformatical search, cloning, and isolation of recombinant Cas9 proteins, testing for the presence of nuclease activity in vitro, and determining the PAM sequence, which is required for recognition of DNA targets, are presented. Potential difficulties that may arise, as well as ways to overcome them, are considered.

The Profile of Post-translational Modifications of Histone H1 in Chromatin of Mouse Embryonic Stem Cells.

Linker histone H1 is one of the main chromatin proteins which plays an important role in organizing eukaryotic DNA into a compact structure. There is data indicating that cell type-specific post-translational modifications of H1 modulate chromatin activity. Here, we compared histone H1 variants from NIH/3T3, mouse embryonic fibroblasts (MEFs), and mouse embryonic stem (ES) cells using matrix-assisted laser desorption/ ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS). We found significant differences in the nature and positions of the post-translational modifications (PTMs) of H1.3-H1.5 variants in ES cells compared to differentiated cells. For instance, methylation of K75 in the H1.2-1.4 variants; methylation of K108, K148, K151, K152 K154, K155, K160, K161, K179, and K185 in H1.1, as well as of K168 in H1.2; phosphorylation of S129, T146, T149, S159, S163, and S180 in H1.1, T180 in H1.2, and T155 in H1.3 were identified exclusively in ES cells. The H1.0 and H1.2 variants in ES cells were characterized by an enhanced acetylation and overall reduced expression levels. Most of the acetylation sites of the H1.0 and H1.2 variants from ES cells were located within their C-terminal tails known to be involved in the stabilization of the condensed chromatin. These data may be used for further studies aimed at analyzing the functional role played by the revealed histone H1 PTMs in the self-renewal and differentiation of pluripotent stem cells.

THE SHORTENED ISOFORM OF a-TUBULIN IS DETECTED IN COMPLEX WITH PROTEASOMES.

The proteasome is a multi-subunit protein complex that serves as a major pathway for intracellular protein degradation playing important functions in various biological processes. By using MALDI-ICR-mass-spectrometry and Western-blot analysis, we have shown the presence of shortened isoform of a-tubulin in complex with the affinity-purified proteasomes from stable cell lines K562 and HEK293.

Post-translational modifications of linker histone H1 variants in mammals.

The covalent modifications of the linker histone H1 and the core histones are thought to play an important role in the control of chromatin functioning. Histone H1 variants from K562 cell line (hH1), mouse (mH1) and calf (cH1) thymi were studied by matrix-activated laser desorption/ionization fourier transform ion cyclotron resonance mass-spectroscopy (MALDI-FT-ICR-MS). The proteomics analysis revealed novel post-translational modifications of the histone H1, such as meK34-mH1.4, meK35-cH1.1, meK35-mH1.1, meK75-hH1.2, meK75-hH1.3, acK26-hH1.4, acK26-hH1.3 and acK17-hH1.1. The comparison of the hH1, mH1 and cH1 proteins has demonstrated that the types and positions of the post-translational modifications of the globular domains of the H1.2-H1.4 variants are very conservative. However, the post-translational modifications of the N- and C-terminal tails of H1.2, H1.3 and H1.4 are different. The differences of post-translational modifications in the N- and C-terminal tails of H1.2, H1.3 and H1.4 likely lead to the differences in DNA-H1 and H1-protein interactions.

RECOVERY OF DIVISION PROCESS IN BACTERIAL CELLS AFTER INDUCTION OF SulA PROTEIN WHICH IS RESPONSIBLE FOR CYTOKINESIS ARREST DURING SOS-RESPONSE.

SOS-response is an important tool of bacteria intended to protect their genome and thereby allow them to survive under adverse conditions. Recently SOS-response is considered to enhance mutagenesis and thus help bacteria acquire antibiotic resistance. Due to high significance of this phenomena it seems to be important to investigate processes that allow bacteria to survive after SOS-response activation. In current work the recovery of division process of Escherichia coli cells after division arrest due to expression of SOS-response protein SulA was studied. Data indicate that cells are able to rapidly restore normal division; also nucleoid occlusion seems to be the main septum positioning mechanism during the process. In the course of recovery FtsZ forms helix-like structures, which then transformate into Z-rings.

Relaxation channels of multi-photon excited xenon clusters.

The relaxation processes of the xenon clusters subjected to multi-photon excitation by laser radiation with quantum energies significantly lower than the thresholds of excitation of atoms and ionization of clusters were studied. Results obtained by means of the photoelectron spectroscopy method showed that desorption processes of excited atoms play a significant role in the decay of two-photon excited xenon clusters. A number of excited states of xenon atoms formed during this process were discovered and identified.

[NEW INFORMATION ABOUT THE STRUCTURES FORMED BY FtsZ PROTEIN IN ESCHERICHIA COLI CELLS DURING DIVISION PROCESS OBTAINED BY SINGLE-MOLECULE LOCALIZATION MICROSCOPY].

FtsZ--a bacterial tubulin homolog--is one of the key bacterial division proteins, forming a contractile Z-ring at the midcell of dividing bacteria. In this work immunofluorescent labeling was used in conjunction with single-molecule localization microscopy (SMLM) to visualize native structures formed by FtsZ protein in Escherichia coli cells. This approach allowed the reorganization of FtsZ structures during cytokinesis to be visualized step-by-step. New data was obtained that support the hypothesis that the Z-ring is a spiral structure that constricts during division, assisting the formation of the septum between daughter cells.

[Mass spectrometric analysis of proteasomes affinity puirified from the human myelogenous leukemia cells K562].

Proteasomes carry out regulated proteolysis of most proteins and thereby play a crucial role in the regulation of different cellular processes. Dissecting subunit composition and post-translational modifications of proteasome is one of the important milestones in understanding their functions and mechanisms of regulation in the cell. To this end a strategy we followed a strategy for affinity purification of proteasomes from human myeloid leukemia cells with subsequent mass spectrometric analysis. Proteasomes were purified from the stable cell line K562 expressing HTBH tag-labeled 20S proteasome subunit ß7 (PSMB4) by non-covalent affinity purification on biotin-avidin beads, followed by elution with TEV protease. We identified all known subunits of the 26S proteasome, as well as PA200 and regulators PA28γ amongst the eluted proteins, using MALDI-ICR mass spectrometry. We have shown that the proteasomes are associated with heat shock proteins, components of the ubiquitin-proteasome system of some cytoskeleton proteins. A number of novel phosphorylation, ubiquitination and N-terminal modification of proteasome subunits were found for 16 proteasome subunits. Our results might be useful for further proteomic studies of proteasomes.

Mass-resolved two-photon and photoelectron spectra of ArXe in the region of Xe* 7p, 6p', 6d.

The two photon resonant, three photon ionization spectra of ArXe were recorded in the spectral region of 88,500-90,100 cm(-1). Seven new molecular band progressions dissociating to ArXe* → Ar(1)S0 + Xe* 7p[1/2]0, Xe* 7p[3/2]2, Xe* 6p'[3/2]2, Xe* 6p'[1/2]1, Xe* 6p'[1/2]0 have been selected and analyzed. The molecular constants for the excited states of ArXe* of these vibrational progressions were determined in the approximation of the anharmonic oscillator, the Morse potential and the Franck-Condon principle. The photoelectron spectra were recorded by several excited electronic-vibrational transitions of ArXe, the dissociation channels of the excited molecules were determined and extra information about the electron structure of the excited molecular states was obtained.

[Comparative structural and functional characteristics of different forms of Saccharomyces cerevisiae red pigment and its synthetic analogue].

Structural and functional characteristics of the yeast red pigment (product of polymerization of N1-(beta-D-ribofuranosyl)-5-aminoimadazole), isolated from adel 1 mutant cells of Saccharomyces cerevisiae, its deribosylated derivatives (obtained by acid hydrolysis) and its synthetic pigment analogue (product of polymerization of N1-methyl-5-aminoimadazole in vitro) has been obtained. Products of in vitro polymerization were identified using mass spectrometry. The ability of these pigments to inhibit amyloid formation using insulin fibrils was compared. The entire compounds studied were able to interact with amyloids and inhibit their growth. Electron and atomic force microscopy revealed a common feature inherent in the insulin fibrils formed in presence of these compounds--they were merged into conglomerates that were more stable and resistant to the effects of ultrasound in comparison with insulin aggregates grown without pigments. We speculate that all these compounds can cause coalescence of fibrils, partially block their loose ends and, thereby, inhibit the attachment of new monomers to growing fibrils.

[The effect of red pigment of Saccharomyces cerevisiae on insulin fibril formation in vitro].

The effect of the yeast red pigment, the result of polymerization of AIR, and of its low molecular weight derivative (presumably devoid of phosphoribosyl moiety) on the formation of amyloid fibrils in vitro was studied. Both the red pigment and its derivative, the result of acid hydrolysis of the original pigment, were shown to diminish the intensity of amyloid bound Thioflavine T fluorescence. Correlation between the decrease of the intensity of Thioflavine T fluorescence and the concentration of both forms of the red pigment was demonstrated. Both forms were also able to compete with Thioflavine T for amyloid fibrils. Electron microscopy permitted to visualize a drop of fibril size in the case of red pigments presence during their formation.

[Experience of vascularized autotransplants use for lower jaw defect substitution after its resection with disarticulation].

To 17 patients after lower jaw resection with disarticulation (on the occasion of neoplasm and traumas) the defects were replaced by vascularized autotransplants of the 2nd radius of pedis and vascularized fibula autotransplants in combination with titanium implants. In all cases positive results were received. The authors consider that microsurgery with vascularized fibula autotransplants in combination with titanium implants was an optimal way to replace lower jaw defects after its resection with disarticulation in TMJ.

[Revelation and identification of laminin in the structure of plasma membrane of ascitic Zajdela hepatoma cells].

Cell dysdifferentiation during neoplastic transformation is a crucial problem of cell biology and oncology. Antigenic diversion of cancer cells is a typical characteristic of dysdifferentiation. It involves the appearance of antigens which are unusual for normal tissue of this type. Components organospecific for membrane proteins of normal kidney were previously found among plasma membrane proteins of hepatocellular rat tumors, rat hepatocytes after carcinogen treatment, and regenerating liver, respectively. In the present work we showed that a protein with mol. weight about 200 kDa reacting with laminin-1 immunoserum is the basic component of plasma membranes of the rat Zajdela hepatoma cells, which is responsive for organospecific anti-kidney immunoserum in Western blot. A mass-spectrometer analysis of trypsin proteolysis fragments was carried out in SDS-PAGE slices containing the investigated component. The analysis showed the presence of beta1, beta2 and alpha4 laminin chains peptides. The component with mol. weight about 180 kDa, found in the Western blot with laminin-1 immunoserum, was also subjected to the mass spectrometer analysis. As a result, a gamma1 laminin chain was found. An increased amount of laminin was revealed in the ascitic liquid and sera of rat with developed Zajdela hepatoma, in comparison with sera of normal rats. In addition, we found the appearance of laminin on the hepatocyte surface on the 4th day after hepatocarcinogen injection (N-diethylnitrosamine, DENA). Thus, for the first time tumor associated antigens were revealed and identified in the structure of plasma membranes of Zajdela hepatoma cells, being specific to rat kidneys. Our results allow to conclude that in the process of carcinogenesis in rat liver laminin synthesis occurs, which is also characteristic of the rat hepatoma Zajdela cells.

[Experience in using complex flaps on microvascular anastomoses in treating with skin cancer on the face and scalp, appearing in scarred areas]. / Opyt ispol'zovaniia slozhnykh loskutov na mikrososudistykh anastomozakh v lechenii bol'nykh rakom kozhi litsa i volosistoi chasti golovy, voznikshim iz rubtsov.

The authors describe 11 patients operated upon for carcinoma of the face skin and hairy part of the head developing from scars of various etiology. The substitution of vast defects which had appeared after dissection of a tumor was performed by means of microsurgical autotransplantation of tissue complexes. The following vascularized autotransplants were used: musculus serratus anterior, scapular and radial grafts. Positive results were obtained in all the cases. The authors consider the microsurgical autotransplantation of tissue complexes to be a necessary component of surgical treatment of carcinoma of the skin and hairy part of the head developed from vast and deep scars.

[Method of plastic replacement of extended laryngopharyngeal and cervical esophagus defects ]. / Sposob plasticheskogo zameshcheniia obshirnykh ziiaiushchikh defektov gortanoglotki i sheinogo otdela pishchevoda.

Filling of extended pharyngoesophagostomas were made in 13 patients operated for advanced laryngeal cancer by means of microsurgical autotransplantation by the forearm radial flap. The latter was created in the form of two cutaneous elements on common fascial base. After transfer of the autotransplant in the recipient's bed, one cutaneous element filled the defect of the laryngopharyngeal and esophageal inner coating, the second filled the defect of the outer coating. This method is reliable, effective, low traumatic, helps to avoid abdominal surgery.

[The organization of microsurgery at a provincial center]. / Organizatsiia mikrokhirurgii v usloviiakh oblastnogo tsentra.

The 5-year experience of microsurgery in regional medical centre is analysed. 1014 operations were performed. Certain managerial items and practical recommendations, necessary for development of microsurgery in regional medical centers are discussed. The importance of a close contract with other surgical services is advocated.

[Reconstructive operations on the head and neck using a microsurgical technic]. / Vosstanovitel'nye operatsiinagolovei shee s primeneniem mikrokhirurgicheskoi tekhniki.

Eighty-one patients with defects and deformations of the head and neck of various origin were examined and treated. Microsurgical autotransplantation of vascularized tissue complexes was carried out in all the cases. Profound preliminary examinations of patients helped objectively define the indications for the use of this method. Special attention was paid to the choice of donor zone. Due to correct selection of a complex graft, good functional and cosmetic results were attained. Despite repeated corrective operations in 16 patients, virtually complete social and communal rehabilitation was attained in 74 (91.4%) patients by using a limited number of donor areas. The authors persuasively demonstrate the advantages of microsurgery in repair of complex and extensive defects on the head and neck.