Mutants of phage bIL67 RuvC with enhanced Holliday junction binding selectivity and resolution symmetry

Viral and bacterial Holliday junction resolvases differ in specificity with the former typically being more promiscuous, acting on a variety of branched DNA substrates, while the latter exclusively targets Holliday junctions. We have determined the crystal structure of a RuvC resolvase from bacteriophage bIL67 to help identify features responsible for DNA branch discrimination. Comparisons between phage and bacterial RuvC structures revealed significant differences in the number and position of positively-charged residues in the outer sides of the junction binding cleft. Substitutions were generated in phage RuvC residues implicated in branch recognition and six were found to confer defects in Holliday junction and replication fork cleavage in vivo. Two mutants, R121A and R124A that flank the DNA binding site were purified and exhibited reduced in vitro binding to fork and linear duplex substrates relative to the wild-type, while retaining the ability to bind X junctions. Crucially, these two variants cleaved Holliday junctions with enhanced specificity and symmetry, a feature more akin to cellular RuvC resolvases. Thus, additional positive charges in the phage RuvC binding site apparently stabilize productive interactions with branched structures other than the canonical Holliday junction, a feature advantageous for viral DNA processing but deleterious for their cellular counterparts

Bioactive Calcium Phosphate Coatings on Metallic Implants

Biocomposites based on bioinert metals or alloys and bioactive calcium phosphate coatings are a promising tendency of the new-generation implants development. In recent years, the approach of regenerative medicine based on the use of biodegradable biomaterials has been priority direction. Such materials are capable of initiating the bone tissue regeneration and replaced by the newly formed bone. The microarc oxidation (MAO) method allows obtaining the bioactive coatings with a porous structure, special functional properties, and modified by the essential elements. During the last decade, the investigations in the field of the nanostructured biocomposites based on bioinert Ti, Zr, Nb and their alloys with a calcium phosphate coatings deposited by the MAO method have been studied in the Institute of Strength Physics and Materials Science SB RAS, Tomsk. In this article the possibility to produce the bioactive coatings with high antibacterial and osseoconductive properties due to the introduction in the coatings of Zn, Cu, Ag, La, Si elements and wollastonite CaSiO[3] was shown. The high hydrophilic and bioresorbed coatings stimulate the processes of osseointegration of the implant into the bone tissue. A promising direction in the field of the medical material science is a development of the metallic implants with good biomechanical compatibility to the bone, such as Ti-Nb alloys with a low elastic modulus that can be classified as biomaterials of the second generation. Zr and its alloys are promising materials for the dentistry and orthopedic surgery due to their high strength and corrosion resistance. Biodegradable Mg alloys are biomaterials of third generation. Such materials can dissolve with a certain speed in human body and excreted from the body thereby excluding the need for reoperation. This article presents the analysis of the study results of bioactive MAO coatings on Ti, Ti-Nb, Zr-Nb and Mg alloys and their promising medical application

Intensification of copper leaching from heaps using biological oxidation

The article presents the results of experiments intended to leach copper from ore heaps of complex mineral composition biochemically. The processing of such heaps is complicated due to the presence of oxidized copper minerals among significant fragments of sulfide minerals and iron-calcium silicates. This factor does not allow to perform standard sulfuric acid leaching effectively without the use of additional oxidation catalysts, or to apply beneficiation methods for that kind of raw materials. Use of A. Ferrooxidans bacteria adapted to the composition of the copper dump, as a bio-catalytic agent, significantly accelerates the leaching process and increases the copper recovery degree into the productive solution

Intensification of copper leaching from heaps using biological oxidation

The article presents the results of experiments intended to leach copper from ore heaps of complex mineral composition biochemically. The processing of such heaps is complicated due to the presence of oxidized copper minerals among significant fragments of sulfide minerals and iron-calcium silicates. This factor does not allow to perform standard sulfuric acid leaching effectively without the use of additional oxidation catalysts, or to apply beneficiation methods for that kind of raw materials. Use of A. Ferrooxidans bacteria adapted to the composition of the copper dump, as a bio-catalytic agent, significantly accelerates the leaching process and increases the copper recovery degree into the productive solution

Light-induced sulfur transport inside single-walled carbon nanotubes

This article belongs to the Section 2D and Carbon Nanomaterials.Filling of single-walled carbon nanotubes (SWCNTs) and extraction of the encapsulated species from their cavities are perspective treatments for tuning the functional properties of SWCNT-based materials. Here, we have investigated sulfur-modified SWCNTs synthesized by the ampoule method. The morphology and chemical states of carbon and sulfur were analyzed by transmission electron microscopy, Raman scattering, thermogravimetric analysis, X-ray photoelectron and near-edge X-ray absorption fine structure spectroscopies. Successful encapsulation of sulfur inside SWCNTs cavities was demonstrated. The peculiarities of interactions of SWCNTs with encapsulated and external sulfur species were analyzed in details. In particular, the donor–acceptor interaction between encapsulated sulfur and host SWCNT is experimentally demonstrated. The sulfur-filled SWCNTs were continuously irradiated in situ with polychromatic photon beam of high intensity. Comparison of X-ray spectra of the samples before and after the treatment revealed sulfur transport from the interior to the surface of SWCNTs bundles, in particular extraction of sulfur from the SWCNT cavity. These results show that the moderate heating of filled nanotubes could be used to de-encapsulate the guest species tuning the local composition, and hence, the functional properties of SWCNT-based materials.This work was supported by the Russian Science Foundation (Project 18-72-00017), the bilateral Program “Russian-Germany Laboratory at BESSY II” in the part of XPS and C K-edge NEXAFS measurements, and shared research center SSTRC on the basis of the Novosibirsk VEPP-4 - VEPP-2000 complex at BINP SB RAS, using equipment supported by project RFMEFI62119X0022 in the part of S K-edge NEXAFS measurements. R.A. acknowledges the support from the Spanish Ministerio de Economia y Competitividad (MAT2016-79776-P, AEI/FEDER, EU), from the European Union’s Horizon 2020 programme under the project “ESTEEM3” (823717) and from the Government of Aragon and the European Social Fund under the project “Construyendo Europa desde Aragon” 2014–2020 (grant number E13_17R, FEDER, EU).Peer reviewe

Research of biochemical gold recovery method using high-arsenic raw materials

This article contains the results of experiments to recover gold from complex mineral raw materials containing more than 15 % arsenic. Laboratory tests showed that standard cyanidation recovers only 26,4 % of gold into the solution. Additional oxidizing reagents used increase the leaching efficiency and enable to recover more than 40 % of gold during subsequent cyanidation. The efficiency has been established for replacement of cyanide with thiourea and thiosulfate solutions. 79,5 %, i.e. the maximum recovery rate, was found in the experiment with preliminary oxidation with T. Ferrooxidans, a bacterial culture, followed by leaching with a thiourea solution

Research of biochemical gold recovery method using high-arsenic raw materials

This article contains the results of experiments to recover gold from complex mineral raw materials containing more than 15 % arsenic. Laboratory tests showed that standard cyanidation recovers only 26,4 % of gold into the solution. Additional oxidizing reagents used increase the leaching efficiency and enable to recover more than 40 % of gold during subsequent cyanidation. The efficiency has been established for replacement of cyanide with thiourea and thiosulfate solutions. 79,5 %, i.e. the maximum recovery rate, was found in the experiment with preliminary oxidation with T. Ferrooxidans, a bacterial culture, followed by leaching with a thiourea solution

H2AX phosphorylation at the sites of DNA double-strand breaks in cultivated mammalian cells and tissues

A sequence variant of histone H2A called H2AX is one of the key components of chromatin involved in DNA damage response induced by different genotoxic stresses. Phosphorylated H2AX (γH2AX) is rapidly concentrated in chromatin domains around DNA double-strand breaks (DSBs) after the action of ionizing radiation or chemical agents and at stalled replication forks during replication stress. γH2AX foci could be easily detected in cell nuclei using immunofluorescence microscopy that allows to use γH2AX as a quantitative marker of DSBs in various applications. H2AX is phosphorylated in situ by ATM, ATR, and DNA-PK kinases that have distinct roles in different pathways of DSB repair. The γH2AX serves as a docking site for the accumulation of DNA repair proteins, and after rejoining of DSBs, it is released from chromatin. The molecular mechanism of γH2AX dephosphorylation is not clear. It is complicated and requires the activity of different proteins including phosphatases and chromatin-remodeling complexes. In this review, we summarize recently published data concerning the mechanisms and kinetics of γH2AX loss in normal cells and tissues as well as in those deficient in ATM, DNA-PK, and DSB repair proteins activity. The results of the latest scientific research of the low-dose irradiation phenomenon are presented including the bystander effect and the adaptive response estimated by γH2AX detection in cells and tissues

Direct observation of DNA threading in flap endonuclease complexes

Maintenance of genome integrity requires that branched nucleic acid molecules are accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in complexes with intact DNA substrates, and products, at resolutions of 1.9–2.2 Å. They reveal single-stranded DNA threading through a hole in the enzyme enclosed by an inverted Vshaped helical arch straddling the active site. Residues lining the hole induce an unusual barb-like conformation in the DNA substrate juxtaposing the scissile phosphate and essential catalytic metal ions. A series of complexes and biochemical analyses show how the substrate’s single-stranded branch approaches, threads through, and finally emerges on the far side of the enzyme. Our studies suggest that substrate recognition involves an unusual “flycasting, thread, bend and barb” mechanis

Radiation-Induced Bystander Effects in Cultured Human Stem Cells

The radiation-induced "bystander effect" (RIBE) was shown to occur in a number of experimental systems both in vitro and in vivo as a result of exposure to ionizing radiation (IR). RIBE manifests itself by intercellular communication from irradiated cells to non-irradiated cells which may cause DNA damage and eventual death in these bystander cells. It is known that human stem cells (hSC) are ultimately involved in numerous crucial biological processes such as embryologic development; maintenance of normal homeostasis; aging; and aging-related pathologies such as cancerogenesis and other diseases. However, very little is known about radiation-induced bystander effect in hSC. To mechanistically interrogate RIBE responses and to gain novel insights into RIBE specifically in hSC compartment, both medium transfer and cell co-culture bystander protocols were employed.Human bone-marrow mesenchymal stem cells (hMSC) and embryonic stem cells (hESC) were irradiated with doses 0.2 Gy, 2 Gy and 10 Gy of X-rays, allowed to recover either for 1 hr or 24 hr. Then conditioned medium was collected and transferred to non-irradiated hSC for time course studies. In addition, irradiated hMSC were labeled with a vital CMRA dye and co-cultured with non-irradiated bystander hMSC. The medium transfer data showed no evidence for RIBE either in hMSC and hESC by the criteria of induction of DNA damage and for apoptotic cell death compared to non-irradiated cells (p>0.05). A lack of robust RIBE was also demonstrated in hMSC co-cultured with irradiated cells (p>0.05).These data indicate that hSC might not be susceptible to damaging effects of RIBE signaling compared to differentiated adult human somatic cells as shown previously. This finding could have profound implications in a field of radiation biology/oncology, in evaluating radiation risk of IR exposures, and for the safety and efficacy of hSC regenerative-based therapies