The identification of mitochondrial DNA variants in glioblastoma multiforme

Background: Mitochondrial DNA (mtDNA) encodes key proteins of the electron transfer chain (ETC), which produces ATP through oxidative phosphorylation (OXPHOS) and is essential for cells to perform specialised functions. Tumor-initiating cells use aerobic glycolysis, a combination of glycolysis and low levels of OXPHOS, to promote rapid cell proliferation and tumor growth. Glioblastoma multiforme (GBM) is an aggressively malignant brain tumor and mitochondria have been proposed to play a vital role in GBM tumorigenesis. Results: Using next generation sequencing and high resolution melt analysis, we identified a large number of mtDNA variants within coding and non-coding regions of GBM cell lines and predicted their disease-causing potential through in silico modeling. The frequency of variants was greatest in the D-loop and origin of light strand replication in non-coding regions. ND6 was the most susceptible coding gene to mutation whilst ND4 had the highest frequency of mutation. Both genes encode subunits of complex I of the ETC. These variants were not detected in unaffected brain samples and many have not been previously reported. Depletion of HSR-GBM1 cells to varying degrees of their mtDNA followed by transplantation into immunedeficient mice resulted in the repopulation of the same variants during tumorigenesis. Likewise, de novo variants identified in other GBM cell lines were also incorporated. Nevertheless, ND4 and ND6 were still the most affected genes. We confirmed the presence of these variants in high grade gliomas. Conclusions: These novel variants contribute to GBM by rendering the ETC. partially dysfunctional. This restricts metabolism to anaerobic glycolysis and promotes cell proliferation

Regularities of the deformed microstructure of ferritic-martensitic steel EP-823 after high-temperature thermomechanical treatment

The features of the microstructure of 12% chromium ferritic-martensitic steel EP-823 near the neck region of samples deformed by tension at T=-70, -40 °C, 20 °C and in the temperature range close to the reactor core operating temperatures of T = 650, 720 °C after high-temperature thermomechanical treatment (HTMT) are investigated. It is shown that at negative and room temperatures plastic deformation leads to curvature and fragmentation of martensitic lamellae, formation of new low-angle boundaries and a significant increase in the dislocations density. Deformation at high temperature contributes to the dynamic recovery, dynamic polygonization and increased sizes of carbide and carbonitride particles (M23C6 and MX type)

Effect of thermomechanical treatment modes on structural-phase states and mechanical properties of metastable austenitic steel

The features of the structural-phase states and mechanical properties of metastable austenitic steel after thermomechanical treatments have been investigated. It is shown that low-temperature and subsequent deformation in the temperature range 300–773 K contributes to the direct (γ → α′)-martensitic transformation. The combination of low-temperature, subsequent warm deformation at 873 K and annealing at 1073 K leads to the direct (γ → α′)- and reverse (α′ → γ)-martensitic transformations. As a result of thermomechanical treatments submicrocrystalline two-phase structural states with high strength properties (σ0.1 ≈ 1160–1350 MPa) are formed

Features of deformation localization in stable austenitic steel under thermomechanical treatment

Features of structural states of Fe-18Cr-14Ni-Mo austenitic steel after thermomechanical treatment, including low-temperature and warm rolling deformation, were investigated by means of transmission electron microscopy. It is shown that mechanical twinning in multiple systems and strain localization bands contribute to grain fragmentation with the formation of the submicrocrystalline austenitic structure. These bands lie in the miсrotwin structure, have high-angle (≈60°–90°, 〈110〉) misorientations of the crystal lattice relative to the matrix and localize significant (up to ≈1) shear strain. In areas of the bands, structural states with high (tens of deg/μm) curvature of the crystal lattice and high local internal stresses are observed. The internal structure of the bands is presented by nanoscale fragments of austenite and α′-martensite. The presence of specific misorientations and fragments of martensite means that the formation mechanism of localized deformation bands are direct plus reverse (γ → α′ → γ) martensitic transformations with the reverse transformation follows by an alternative path. These structural states provide high strength properties of steel: the yield strength is up to 1150 MPa

Macromolecular mimicry in protein biosynthesis

Elongation factor Tu (EF-Tu) is a G-protein which, in its active GTP conformation, protects and carries aminoacylated tRNAs (aa-tRNAs) to the ribosome during protein biosynthesis. EF-Tu consists of three structural domains of which the N-terminal domain consists of two special regions (switch I and switch II) which are structurally dependent on the type of the bound nucleotide. Structural studies of the complete functional cycle of EF-Tu reveal that it undergoes rather spectacular conformational changes when activated from the EF-Tu·GDP form to the EF-Tu·GTP form. In its active form, EF-Tu·GTP without much further structural change interacts with aa-tRNAs in the so-called ternary complex. The conformational changes of EF-Tu involve rearrangements of the secondary structures of both the switch I and switch II regions. As the switch II region forms part of the interface between domains 1 and 3, its structural rearrangement results in a very large change of the position of domain 1 relative to domains 2 and 3. The overall shape of the ternary complex is surprisingly similar to the overall shape of elongation factor G (EF-G). Thus, three domains of the protein EF-G seem to mimic the tRNA part of the ternary complex. This macromolecular mimicry has profound implications for the function of the elongation factors on the ribosome

Elucidation of the substrate binding site of Siah ubiquitin ligase

The Siah family of RING proteins function as ubiquitin ligase components, contributing to the degradation of multiple targets involved in cell growth, differentiation, angiogenesis, oncogenesis, and inflammation. Previously, a binding motif (degron) was recognized in many of the Siah degradation targets, suggesting that Siah itself may facilitate substrate recognition. We report the crystal structure of the Siah in complex with a peptide containing the degron motif. Binding is within a groove formed in part by the zinc fingers and the first two ß strands of the TRAF-C domain of Siah. We show that residues in the degron, previously described to facilitate binding to Siah, interact with the protein. Mutagenesis of Siah at sites of interaction also abrogates both in vitro peptide binding and destabilization of a known Siah target

The features of microstructure and mechanical properties of austenitic steel after direct and reverse martensitic transformations

The features of structural states of metastable austenitic steel after thermomechanical treatments, including low-temperature deformation, warm deformation and subsequent annealing are investigated. It is shown that under these conditions the direct (γ → α′) and reverse (α′ → γ) martensitic transformations occur and submicrocrystalline structural states are formed. The proposed thermomechanical treatment allows varying the strength and plastic properties of austenitic steel in a wide range. The strength of steel in submicrocrystalline state is 4–6 times higher than its original value

Effect of multistage high temperature thermomechanical treatment on the microstructure and mechanical properties of austenitic reactor steel

The deformation microstructures formed by novel multistage high-temperature thermomechanical treatment (HTMT) and their effect on the mechanical properties of austenitic reactor steel are investigated. It is shown that HTMT with plastic deformation at the temperature decreasing in each stage (1100, 900, and 600 C with a total strain degree of e = 2) is an effective method for refining the grain structure and increasing the strength of the reactor steel. The structural features of grains, grain boundaries and defective substructure of the steel are studied in two sections (in planes perpendicular to the transverse direction and perpendicular to the normal direction) by Scanning Electron Microscopy with Electron Back-Scatter Diffraction (SEM EBSD) and Transmission Electron Microscopy (TEM). After the multistage HTMT, a fragmented structure is formed with grains elongated along the rolling direction and flattened in the rolling plane. The average grain size decreases from 19.3 m (for the state after solution treatment) to 1.8 m. A high density of low-angle boundaries (up to 80%) is found inside deformed grains. An additional cold deformation (e = 0.3) after the multistage HTMT promotes mechanical twinning within fragmented grains and subgrains. The resulting structural states provide high strength properties of steel: the yield strength increases up to 910 MPa (at 20 C) and up to 580 MPa (at 650 C), which is 4.6 and 6.1 times higher than that in the state after solution treatment (ST), respectively. The formation of deformed substructure and the influence of dynamic strain aging at an elevated tensile temperature on the mechanical properties of the steel are discussed. Based on the results obtained, the multistage HTMT used in this study can be applied for increasing the strength of austenitic steels

Microstructure and mechanical properties of heat-resistant 12 % Cr ferritic-martensitic steel EK-181 after thermomechanical treatment

The effect of high-temperature thermomechanical treatment (TMT) with the deformation in the austenitic region on the features of microstructure, phase transformations and mechanical properties of low-activation 12% Cr ferritic-martensitic steel EK-181 is investigated. It is established, that directly after thermomechanical treatment (without tempering) the sizes and density of V(CN) particles are comparable with those after a traditional heat treatment (air quenching and tempering at 720°C, 3 h), where these particles are formed only during tempering. It causes the increasing of the yield strength of the steel up to ≈1450 MPa at room temperature and up to ≈430 MPa at the test temperature T = 650°C. The potential of microstructure modification by this treatment aimed at improving heat resistance of steel is discussed

Differential regulation by AMP and ADP of AMPK complexes containing different γ subunit isoforms

The γ subunits of heterotrimeric AMPK complexes contain the binding sites for the regulatory adenine nucleotides AMP, ADP and ATP. We addressed whether complexes containing different γ isoforms display different responses to adenine nucleotides by generating cells stably expressing FLAG-tagged versions of the γ 1, γ 2 or γ 3 isoform. When assayed at a physiological ATP concentration (5 mM), γ 1- and γ 2-containing complexes were allosterically activated almost 10-fold by AMP, with EC50 values one to two orders of magnitude lower than the ATP concentration. By contrast, γ 3 complexes were barely activated by AMP under these conditions, although we did observe some activation at lower ATP concentrations. Despite this, all three complexes were activated, due to increased Thr172 phosphorylation, when cells were incubated with mitochondrial inhibitors that increase cellular AMP. With γ 1 complexes, activation and Thr172 phosphorylation induced by the upstream kinase LKB1 [liver kinase B1; but not calmodulin-dependent kinase kinase (CaMKKβ)] in cell-free assays was markedly promoted by AMP and, to a smaller extent and less potently, by ADP. However, effects of AMP or ADP on activation and phosphorylation of the γ 2 and γ 3 complexes were small or insignificant. Binding of AMP or ADP protected all three γ subunit complexes against inactivation by Thr172 dephosphorylation; with γ 2 complexes, ADP had similar potency to AMP, but with γ 1 and γ 3 complexes, ADP was less potent than AMP. Thus, AMPK complexes containing different γ subunit isoforms respond differently to changes in AMP, ADP or ATP. These differences may tune the responses of the isoforms to fit their differing physiological roles