Cenozoic intracontinental dextral motion in the Okhotsk-Japan Sea Region

Laurent Jolivet est Professeur à l'Université d'Orléans au 1er Septembre 2009International audienceA right-lateral shear zone trending northerly along more than 2000 km is recognized from central Japan to northern Sakhalin. It was active mainly during the Neogene and has accommodated several hundreds of kilometers of displacement. The whole structure of Sakhalin is built on this shear zone. En échelon sigmoidal folds and thrusts, en échelon narrow Miocene basins, and a major discontinuity which is observed along more than 600 km, the Tym-Poronaisk fault, characterize the deformation there. In Hokkaido, en échelon folds and thrusts and a ductile shear zone with high-temperature metamorphism constitute the southern extension of this transpressional shear zone. It continues to the south as a zone of transtensional deformation along the eastern margin of Japan Sea, as en échelon basins and dextral transfer faults observed as far south as Noto peninsula and Yatsuo basin. The style of the shear zone thus evolves from transpressional in the north far from the subduction zone, to transtensional in the south in the back-arc region. Strike-slip motion along this shear zone was primarily responsible for the dextral pull-apart opening of Japan Sea during the early and middle Miocene. Dextral motion is still active in the north along the Tym-Poronaisk fault in Sakhalin as well as on the continental margin of Japan Sea (Korea and Asia mainland). Active E-W compression replaced the dextral motion along the eastern margin of Japan Sea in late Miocene time, and incipient subduction began in the early Quaternary

Neogene strike-slip faulting in Sakhalin and the Japan Sea opening

Laurent Jolivet est Professeur à l'Université d'Orléans au 1er Septembre 2009International audienceWe describe structural data from a 2000 km N-S dextral strike-slip zone extending from northern Sakhalin to the southeast corner of the Japan Sea. Satellite images, field data, and focal mechanisms of earthquakes in Sakhalin are included in the interpretation. Since Miocene time the deformation in Sakhalin has been taken up by N-S dextral strike-slip faults with a reverse component and associated en e'chelon folds. Narrow en échelon Neogene basins were formed along strike-sup faults and were later folded in a second stage of deformation. We propose a model of basin formation along extension al faults delimitating dominos between two major strike-slip faults, and subsequent counterclockwise rotation of the dominos in a dextral transpressional regime, basins becoming progressively oblique to the direction of maximum horizontal compression and undergoing shortening. The association of both dextral and compressional focal mechanisms of earthquakes indicates that the same transpressional regime still prevails today in Sakhalin. We present fault set measurements undertaken in Noto Peninsula and Yatsuo Basin at the southern end of the Sakhalin-East Japan Sea strike-slip zone. Early and middle Miocene formations recorded the same transtensional regime as observed along the west coast of NE Honshu. During the early and middle Miocene the strike-slip regime was transpressional to the north in Sakhalin and Hokkaido, and transtensional to the south along the west coast of NE Honshu as far as Noto Peninsula and Yatsuo basin. Dextral motion accommodated the opening of the Japan Sea as a pull-apart basin, with the Tsushima fault to the west. The opening of the Japan Sea ceased at the end of the middle Miocene when transtension started to change to E-W compression in the Japan arc. Subduction of the Japan Sea lithosphere under the Japan arc started 1.8 Ma ago. The evolution of the stress regime from transtensional to compressional in the southern part of the strike-slip zone is related to the inception of the subduction of the young Philippine Sea Plate lithosphere under the Japan arc during the late Miocene. Subduction related extension is a necessary condition for the opening of the Japan Sea. Two possible mechanisms can account for dextral shear in this area: (1) counterclockwise rotation of crustal blocks due to the collision of India with Asia, (2) extrusion of the Okhotsk Sea block squeezed between the North America and Eurasia plates

DETERMINING REFERENCE RANGES FOR TREC AND KREC ASSAYS IN IMMUNE DEFICIENCY SCREENING OF NEWBORNS IN RUSSIAN FEDERATION

In this work, we used a reference population of newborns and sampled dried blood spots on Guthrie cards of 2,739 individual samples to determine the reference intervals for TRECs and KRECs values, in order to diagnose primary immunodeficiency by means of neonatal screening. The median absolute values for TRECs and KRECs were 195 (CI95%: 185-206) and 185 (CI95%: 176-197) copies per μl, respectively; the normalized value for TRECs was 2780 (CI95%: 2690-2840), and for KRECs, 2790 (CI95%: 2700-2900) copies per 2 × 105 copies of the albumin gene or 105 cells. The reference interval was calculated for 99 and 99.9 percentiles of total TRECs and KRECs individual values. Due to asymmetric distribution of data, the outliers were filtered off, using the Tukey’s criterion applied after logarithmic transformation of the data. When analyzing absolute values for TREC/KREC (per μL of blood), no “drop-down” TRECs values were identified; for KRECs, 18 experimental values were excluded from further analysis (from 9.8 to 13.5). The outlying values were not identified among the normalized values of TRECs/KRECs. The obtained reference values for TRECs and KRECs (at the 0.1 percentile level) were, respectively, 458 and 32 per 105 cells, or 23 and 17 per μl of blood samples from neonates

The attachment of a DNA-binding Sso7d-like protein improves processivity and resistance to inhibitors of M-MuLV reverse transcriptase

Reverse transcriptases (RTs) are a standard tool in both fundamental studies and diagnostics. RTs should possess elevated temperature optimum, high thermal stability, processivity and tolerance to contaminants. Here, we constructed a set of chimeric RTs, based on the combination of the Moloney murine leukaemia virus (M-MuLV) RT and either of two DNA-binding domains: the DNA-binding domain of the DNA ligase from Pyrococcus abyssi or the DNA-binding Sto7d protein from Sulfolobus tokodaii. The processivity and efficiency of cDNA synthesis of the chimeric RT with Sto7d at the C-end are increased several fold. The attachment of Sto7d enhances the tolerance of M-MuLV RT to the most common amplification inhibitors: NaCl, urea, guanidinium chloride, formamide, components of human whole blood and human blood plasma. Thus, fusing M-MuLV RT with an additional domain results in more robust and efficient RTs

Detection of SARS-CoV-2 RNA by a Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Melting Curves Analysis.

Loop-mediated isothermal amplification (LAMP) is a method of nucleic acid amplification that is more stable and resistant to DNA amplification inhibitors than conventional PCR. LAMP multiplexing with reverse transcription allows for the single-tube amplification of several RNA fragments, including an internal control sample, which provides the option of controlling all analytical steps. We developed a method of SARS-CoV-2 viral RNA detection based on multiplex reverse-transcription LAMP, with single-tube qualitative analysis of SARS-CoV-2 RNA and MS2 phage used as a control RNA. The multiplexing is based on the differences in characteristic melting peaks generated during the amplification process. The developed technique detects at least 20 copies of SARS-CoV-2 RNA per reaction on a background of 12,000 MS2 RNA copies. The total time of analysis does not exceed 40 min. The method validation, performed on 125 clinical samples of patients' nasal swabs, showed a 97.6% concordance rate with the results of real-time (RT)-PCR assays. The developed multiplexed LAMP can be employed as an alternative to PCR in diagnostic practice to save personnel and equipment time

Bst polymerase — a humble relative of Taq polymerase

DNA polymerases are a superfamily of enzymes synthesizing DNA using DNA as a template. They are essential for nucleic acid metabolism and for DNA replication and repair. Modern biotechnology and molecular diagnostics rely heavily on DNA polymerases in analyzing nucleic acids. Among a variety of discovered DNA polymerases, Bst polymerase, a large fragment of DNA polymerase I from Geobacillus stearothermophilus, is one of the most commonly used but is not as well studied as Taq polymerase. The ability of Bst polymerase to displace an upstream DNA strand during synthesis, coupled with its moderate thermal stability, has provided the basis for several isothermal DNA amplification methods, including LAMP, WGA, RCA, and many others. Bst polymerase is one of the key components defining the robustness and analytical characteristics of diagnostic test systems based on isothermal amplification. Here, we present an overview of the biochemical and structural features of Bst polymerase and provide information on its mutated analogs

A Novel Thermostable and Processive Reverse Transcriptase from a Group II Intron of Anoxybacillus flavithermus

Reverse transcriptases (RTs) are a family of enzymes that synthesize DNA using an RNA template and are involved in retrovirus propagation and telomere lengthening. In vitro, RTs are widely applied in various methods, including RNA-seq, RT-PCR, and RT-LAMP. Thermostable RTs from bacterial group II introns are promising tools for biotechnology due to their higher thermostability, fidelity, and processivity compared to commonly used M-MuLV RT and its mutants. However, the diversity of group II intron-encoded RTs is still understudied. In this work, we biochemically characterized a novel RT from a thermophilic bacterium, Anoxybacillus flavithermus, which was isolated from a hot spring in New Zealand and has an optimal growth temperature of around 60 °C. The cloned RT, named Afl RT, retained approximately 40% of the specific activity after a 45 min incubation at 50 °C. The optimal pH was 8.5, the optimal temperature was between 45 and 50 °C, and Mn2+ ions were found to be an optimal cofactor. The processivity analysis with MS2 phage gRNA (3569 b) demonstrated that Afl RT elongated fully up to 36% of the template molecules. In reverse transcription and RT-qLAMP, the enzyme allowed up to 10 copies of MS2 phage genomic RNA to be detected per reaction. Thus, Afl RT holds great potential for a variety of practical applications that require the use of thermostable and processive RTs

Optimization of Conditions for Production of Soluble E. coli Poly(A)-Polymerase for Biotechnological Applications

Poly(A) polymerase (PAP 1) from Escherichia coli is the primary enzyme responsible for synthesizing poly(A) tails on RNA molecules, signaling RNA degradation in bacterial cells. In vitro, PAP 1 is used to prepare libraries for RNAseq and to produce mRNA vaccines. However, E. coli PAP 1’s toxicity and instability in low-salt buffers complicate its expression and purification. Here, we optimized the conditions for the production of recombinant PAP 1. For that, E. coli PAP 1 was expressed in seven E. coli strains with different origins and genetic backgrounds, followed by assessment of the overall protein yield, solubility, and enzymatic activity. Among the tested strains, BL21 (DE3) pLysS achieved the best balance of cell density, total PAP 1 yield, solubility, and specific activity. Rosetta 2 (DE3) and Rosetta Blue (DE3) hosting the pRARE plasmid exhibited the lowest solubility, likely due to excessive translation efficiency. Higher induction temperatures (>18 °C) exacerbated PAP 1’s insolubility. Interestingly, PAP 1 accumulation correlated with an increase in the plasmid copy number encoding the enzyme, indicating its potential utility as a surrogate marker of PAP 1 activity. These findings provide insights into optimizing E. coli PAP 1 production for biotechnological applications