Two superconducting states with broken time-reversal symmetry in FeSe1-xSx.

Iron-chalcogenide superconductors FeSe1-xSx possess unique electronic properties such as nonmagnetic nematic order and its quantum critical point. The nature of superconductivity with such nematicity is important for understanding the mechanism of unconventional superconductivity. A recent theory suggested the possible emergence of a fundamentally new class of superconductivity with the so-called Bogoliubov Fermi surfaces (BFSs) in this system. However, such an ultranodal pair state requires broken time-reversal symmetry (TRS) in the superconducting state, which has not been observed experimentally. Here, we report muon spin relaxation (µSR) measurements in FeSe1-xSx superconductors for 0 ≤ x ≤ 0.22 covering both orthorhombic (nematic) and tetragonal phases. We find that the zero-field muon relaxation rate is enhanced below the superconducting transition temperature Tc for all compositions, indicating that the superconducting state breaks TRS both in the nematic and tetragonal phases. Moreover, the transverse-field µSR measurements reveal that the superfluid density shows an unexpected and substantial reduction in the tetragonal phase (x > 0.17). This implies that a significant fraction of electrons remain unpaired in the zero-temperature limit, which cannot be explained by the known unconventional superconducting states with point or line nodes. The TRS breaking and the suppressed superfluid density in the tetragonal phase, together with the reported enhanced zero-energy excitations, are consistent with the ultranodal pair state with BFSs. The present results reveal two different superconducting states with broken TRS separated by the nematic critical point in FeSe1-xSx, which calls for the theory of microscopic origins that account for the relation between nematicity and superconductivity.

Long-read sequencing identifies an SVA_D retrotransposon insertion deep within the intron of ATP7A as a novel cause of occipital horn syndrome.

BACKGROUND: SINE-VNTR-Alu (SVA) retrotransposons move from one genomic location to another in a 'copy-and-paste' manner. They continue to move actively and cause monogenic diseases through various mechanisms. Currently, disease-causing SVA retrotransposons are classified into human-specific young SVA_E or SVA_F subfamilies. In this study, we identified an evolutionarily old SVA_D retrotransposon as a novel cause of occipital horn syndrome (OHS). OHS is an X-linked, copper metabolism disorder caused by dysfunction of the copper transporter, ATP7A. METHODS: We investigated a 16-year-old boy with OHS whose pathogenic variant could not be detected via routine molecular genetic analyses. RESULTS: A 2.8 kb insertion was detected deep within the intron of the patient's ATP7A gene. This insertion caused aberrant mRNA splicing activated by a new donor splice site located within it. Long-read circular consensus sequencing enabled us to accurately read the entire insertion sequence, which contained highly repetitive and GC-rich segments. Consequently, the insertion was identified as an SVA_D retrotransposon. Antisense oligonucleotides (AOs) targeting the new splice site restored the expression of normal transcripts and functional ATP7A proteins. AO treatment alleviated excessive accumulation of copper in patient fibroblasts in a dose-dependent manner. Pedigree analysis revealed that the retrotransposon had moved into the OHS-causing position two generations ago. CONCLUSION: This is the first report of a human monogenic disease caused by the SVA_D retrotransposon. The fact that the evolutionarily old SVA_D is still actively transposed, leading to increased copy numbers may make a notable impact on rare genetic disease research.

Daidara: A gigantic Gypsy LTR retrotransposon lineage in the springtail Allacma fusca genome.

Long terminal repeat (LTR) retrotransposons are the major contributor to genome size expansion, as in the cases of the maize genome or the axolotl genome. Despite their impact on the genome size, the length of each retrotransposon is limited, compared to DNA transposons, which sometimes exceed over 100 kb. The longest LTR retrotransposon known to date is Burro-1 from the planarian Schmidtea medierranea, which is around 35.7 kb long. Here through bioinformatics analysis, a new lineage of gigantic LTR retrotransposons, designated Daidara, is reported from the springtail Allacma fusca genome. Their entire length (25-33 kb) rivals Burro families, while their LTRs are shorter than 1.5 kb, in contrast to other gigantic LTR retrotransposon lineages Burro and Ogre, whose LTRs are around 5 kb long. Daidara encodes three core proteins corresponding to gag, pol, and an additional protein of unknown function. The phylogenetic analysis supports the independent gigantification of Daidara from Burro or Ogre.

Producing Conventional and Transient Amino(mercapto)methyl Radicals by Addition of Muonium to a Crystalline Thioformamide (Mes*NHCH=S).

Muonium (Mu=µ+e-) is composed of a muon of light isotope of proton (µ+) and electron (e-) and can be used as a light surrogate for a hydrogen atom. In this paper, we investigated addition of muonium to a newly synthesized Mes*-substituted thioformamide (Mes*NHCH=S, Mes*=2,4,6-tBu3C6H2). Transverse-field muon spin rotation (TF-µSR) of a solution sample of the thioformamide confirmed addition of muonium to the sulfur atom leading to the corresponding C-centered radical [Mes*NHC(H)⋅-SMu]. Density functional theory (DFT) calculations assigned a conventional amino(mercapto)methyl radical, in which both nitrogen and carbon were slightly pyramidalized, and the calculated muon hyperfine coupling constant (hfcc) including the muon isotope effect was compatible with the experimentally determined parameter. However, the muon level-crossing resonance (µLCR) spectrum of an anisotropic crystalline sample indicated two paramagnetic species, and the major product showed the considerably larger muon hfcc compared with the conventional structure of the amino(mercapto)methyl radical. The unusual transient muoniated thioformamide with the larger muon hfcc that showed rapid relaxation could be only explained by a transient structure including planarization of the nitrogen and carbon atoms in Mes*NHC(H)⋅-SMu.

Increase in the solubility of uvsY using a site saturation mutagenesis library for application in a lyophilized reagent for recombinase polymerase amplification.

BACKGROUND: Recombinase uvsY from bacteriophage T4, along with uvsX, is a key enzyme for recombinase polymerase amplification (RPA), which is used to amplify a target DNA sequence at a constant temperature. uvsY, though essential, poses solubility challenges, complicating the lyophilization of RPA reagents. This study aimed to enhance uvsY solubility. METHODS: Our hypothesis centered on the C-terminal region of uvsY influencing solubility. To test this, we generated a site-saturation mutagenesis library for amino acid residues Lys91-Glu134 of the N-terminal (His)6-tagged uvsY. RESULTS: Screening 480 clones identified A116H as the variant with superior solubility. Lyophilized RPA reagents featuring the uvsY variant A116H demonstrated enhanced performance compared to those with wild-type uvsY. CONCLUSIONS: The uvsY variant A116H emerges as an appealing choice for RPA applications, offering improved solubility and heightened lyophilization feasibility.

Difluoromethylborates and Muonium for the Study of Isonitrile Insertion Affording Phenanthridines via Imidoyl Radicals.

The 6-(difluoromethyl)phenanthridine unit is a highly attractive fluoroalkyl-substituted planar nitrogen heterocycle in pharmaceutical and agrochemical research. In this paper, we report that difluoromethylborates can be used as a source of difluoromethyl radicals for isonitrile insertion, leading to 6-(difluoromethyl)phenanthridines. Tuning the aryl substituents in the difluoromethylborates and oxidizing reagents enabled the synthesis of 6-(difluoromethyl)phenanthridines through the generation of difluoromethyl radical and spontaneous intramolecular cyclization of the CF2H-imidoyl radical intermediates. The presence of difluoromethyl radicals was experimentally confirmed, and the reaction mechanisms including imidoyl radical and prompt cyclization reactions could be supported theoretically. Furthermore, we obtained valuable information about the imidoyl radical intermediate by performing transverse-field muon spin rotation (TF-µSR) measurements of 2-isocyano-4'-methoxy-1,1'-biphenyl and using density functional theory (DFT) calculations to interpret the spectra. Muonium, a simple free radical, preferentially adds to the carbon atom of the isonitrile unit, yielding the corresponding imidoyl radical. The temperature dependence of the muon hyperfine coupling constant and the spin relaxation of the muoniated radical signal are compatible with the intramolecular cyclization of biaryl-substituted imidoyl radicals on the µs time scale.

Modified uvsY by N-terminal hexahistidine tag addition enhances efficiency of recombinase polymerase amplification to detect SARS-CoV-2 DNA.

BACKGROUND: Recombinase (uvsY and uvsX) from bacteriophage T4 is a key enzyme for recombinase polymerase amplification (RPA) that amplifies a target DNA sequence at a constant temperature with a single-stranded DNA-binding protein and a strand-displacing polymerase. The present study was conducted to examine the effects of the N- and C-terminal tags of uvsY on its function in RPA to detect SARS-CoV-2 DNA. METHODS: Untagged uvsY (uvsY-Δhis), N-terminal tagged uvsY (uvsY-Nhis), C-terminal tagged uvsY (uvsY-Chis), and N- and C-terminal tagged uvsY (uvsY-NChis) were expressed in Escherichia coli and purified. RPA reaction was carried out with the in vitro synthesized standard DNA at 41 °C. The amplified products were separated on agarose gels. RESULTS: The minimal initial copy numbers of standard DNA from which the amplified products were observed were 6 × 105, 60, 600, and 600 copies for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The minimal reaction time at which the amplified products were observed were 20, 20, 30, and 20 min for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The RPA with uvsY-Nhis exhibited clearer bands than that with either of other three uvsYs. CONCLUSIONS: The reaction efficiency of RPA with uvsY-Nhis was the highest, suggesting that uvsY-Nhis is suitable for use in RPA.

Optimization of reaction condition of recombinase polymerase amplification to detect SARS-CoV-2 DNA and RNA using a statistical method.

Recombinase polymerase amplification (RPA) is an isothermal reaction that amplifies a target DNA sequence with a recombinase, a single-stranded DNA-binding protein (SSB), and a strand-displacing DNA polymerase. In this study, we optimized the reaction conditions of RPA to detect SARS-CoV-2 DNA and RNA using a statistical method to enhance the sensitivity. In vitro synthesized SARS-CoV-2 DNA and RNA were used as targets. After evaluating the concentration of each component, the uvsY, gp32, and ATP concentrations appeared to be rate-determining factors. In particular, the balance between the binding and dissociation of uvsX and DNA primer was precisely adjusted. Under the optimized condition, 60 copies of the target DNA were specifically detected. Detection of 60 copies of RNA was also achieved. Our results prove the fabrication flexibility of RPA reagents, leading to an expansion of the use of RPA in various fields.

Insight into the collagen-degrading activity of a serine protease in the latex of Ficus carica cultivar Masui Dauphine.

Ficus carica produces, in addition to the cysteine protease ficin, a serine protease. Earlier study on a serine protease from F. carica cultivar Brown Turkey showed that it specifically degraded collagen. In this study, we characterized the collagenolytic activity of a serine protease in the latex of F. carica cultivar Masui Dauphine. The serine protease degraded denatured, but not undenatured, acid-solubilized type I collagen. It also degraded bovine serum albumin, while the collagenase from Clostridium histolyticum did not. These results indicated that the serine protease in Masui Dauphine is not collagen-specific. The protease was purified to homogeneity by two-dimensional gel electrophoresis, and its partial amino acid sequence was determined by liquid chromatography-tandem mass spectrometry. BLAST searches against the Viridiplantae (green plants) genome database revealed that the serine protease was a subtilisin-like protease. Our results contrast with the results of the earlier study stating that the serine protease from F. carica is collagen-specific.

Insight into the mechanism of thermostabilization of GH10 xylanase from Bacillus sp. strain TAR-1 by the mutation of S92 to E.

The mechanism of thermostabilization of GH10 xylanase, XynR, from Bacillus sp. strain TAR-1 by the mutation of S92 to E was investigated. Thermodynamic analysis revealed that thermostabilization was driven by the decrease in entropy change of activation for thermal inactivation. Crystallographic analysis suggested that this mutation suppressed the fluctuation of the amino acid residues at position 92-95.

Muonium Addition to a peri-Trifluoromethylated 9-Phosphaanthracene Producing a High-Energy Paramagnetic π-Conjugated Fused Heterocycle.

In this communication, we report muon spin rotation/resonance (µSR) studies for understanding radical reactivity of 10-mesityl-1,8-bis(trifluoromethyl)-9-phosphaanthracene. Transverse-field muon spin rotation (TF-µSR) and muon avoided level-crossing resonance (µLCR) measurements successfully visualized a paramagnetic species produced by regioselective addition of muonium (Mu) to the skeletal phosphorus atom. Density functional theory (DFT) calculations for the P-muoniation product suggested two possible isomers. Whereas the most stable isomer including the envelope-type phosphorus heterocycle shows considerably different hyperfine coupling constants (hfcs) from those of the TF-µSR and µLCR, the metastable structure accompanying the almost planar tricyclic π-conjugated skeleton could simulate the experimentally determined hfcs. The metastable planar π-conjugated paramagnetic tricyclic-fused skeleton is promoted by the larger zero-point energy due to the light muon (µ+ ), one ninth of the proton mass.

Kinetic analysis of inhibition of α-glucosidase by leaf powder from Morus australis and its component iminosugars.

Mulberry leaves contain iminosugars, such as 1-deoxynojirimycin (1-DNJ), fagomine, and 2-O-α-D-galactopyranosyl deoxynojirimycin (GAL-DNJ) that inhibit α-glucosidase. In this study, we quantified iminosugars in Morus australis leaves and made the kinetic analysis in the hydrolysis of maltose by α-glucosidase. By LC-MS/MS, the concentrations of 1-DNJ, fagomine, and GAL-DNJ in the powdered leaves were 4.0, 0.46, and 2.5 mg/g, respectively, and those in the roasted ones were 1.0, 0.24, and 0.73 mg/g, respectively, suggesting that the roasting process degraded iminosugars. Steady-state kinetic analysis revealed that the powdered and roasted leaves exhibited competitive inhibition. At pH 6.0 at 37ºC, the IC50 values of the extracts from the boiled powdered or roasted leaves were 0.36 and 1.1 mg/mL, respectively. At the same condition, the IC50 values of 1-DNJ, fagomine, and GAL-DNJ were 0.70 µg/mL, 0.18 mg/mL, and 2.9 mg/mL, respectively. These results suggested that in M. australis, 1-DNJ is a major inhibitor of α-glucosidase. ABBREVIATIONS: 1-DNJ: 1-deoxynojirimycin; GAL-DNJ: 2-O-α-D-galactopyranosyl-DNJ.

Increase in the thermostability of Bacillus sp. strain TAR-1 xylanase using a site saturation mutagenesis library.

Site saturation mutagenesis library is a recently developed technique, in which any one out of all amino acid residues in a target region is substituted into other 19 amino acid residues. In this study, we used this technique to increase the thermostability of a GH10 xylanase, XynR, from Bacillus sp. strain TAR-1. We hypothesized that the substrate binding region of XynR is flexible, and that the thermostability of XynR will increase if the flexibility of the substrate binding region is decreased without impairing the substrate binding ability. Site saturation mutagenesis libraries of amino acid residues Tyr43-Lys115 and Ala300-Asn325 of XynR were constructed. By screening 480 clones, S92E was selected as the most thermostable one, exhibiting the residual activity of 80% after heat treatment at 80°C for 15 min in the hydrolysis of Remazol Brilliant Blue-xylan. Our results suggest that this strategy is effective for stabilization of GH10 xylanase. ABBREVIATIONS: DNS: 3,5-dinitrosalicylic acid; RBB-xylan: Remazol Brilliant Blue-xylan.

Observation of a Metastable P-Heterocyclic Radical by Muonium Addition to a 1,3-Diphosphacyclobutane-2,4-diyl.

A 1,3-diphosphacyclobutane-2,4-diyl contains a unique unsaturated cyclic unit, and the presence of radical-type centers have been expected as a source of functionality. This study demonstrates that the P-heterocyclic singlet biradical captures muonium (Mu=[µ+ e- ]), the light isotope of a hydrogen radical, to generate an observable P-heterocyclic paramagnetic species. Investigation of a powder sample of 2,4-bis(2,4,6-tri-t-butylphenyl)-1-t-butyl-3-benzyl-1,3-diphosphacyclobutane-2,4-diyl using muon (avoided) level-crossing resonance (µLCR) spectroscopy revealed that muonium adds to the cyclic P2 C2 unit. The muon hyperfine coupling constant (Aµ ) indicated that the phosphorus atom bearing the t-butyl group trapped muonium to provide a metastable P-heterocyclic radical involving the ylidic MuP(<)=C moiety. The observed regioselective muonium addition correlates the canonical formula of 1,3-diphosphacyclobutane-2,4-diyl.

Population Evolution of Helicobacter pylori through Diversification in DNA Methylation and Interstrain Sequence Homogenization.

Decoding of closely related genomes is now revealing the process of population evolution. In bacteria, population divergence appears associated with a unique set of sequence-specific epigenetic DNA methylation systems, often within restriction-modification (RM) systems. They might define a unique gene expression pattern and limit genetic flux between lineages in population divergence. We addressed the contribution of methylation systems to population diversification in panmictic bacterial species, Helicobacter pylori, which shows an interconnected population structure through frequent mutual recombination. We analyzed complete genome sequences of 28 strains collected in Fukui, Japan. Their nucleotide sequences are closely related although fine-scale analyses revealed two subgroups likely reflecting human subpopulations. Their sequences are tightly connected by homologous recombination. Our extensive analysis of RM systems revealed an extreme variability in DNA methyltransferases, especially in their target recognition domains. Their diversity was, however, not immediately related to the genome sequence diversity, except for very closely related strains. An interesting exception is a hybrid strain, which likely has conserved the methylation gene repertoire from one parent but diversified in sequence by massive acquisition of fragmentary DNA sequences from the other parent. Our results demonstrate how a bacterial population can be extremely divergent in epigenetics and yet homogenized in sequence.

Next-generation sequencing-based analysis of reverse transcriptase fidelity.

In this study, we devised a simple and rapid method to analyze fidelity of reverse transcriptase (RT) using next-generation sequencing (NGS). The method comprises a cDNA synthesis reaction from standard RNA with a primer containing a tag of 14 randomized bases and the RT to be tested, PCR using high-fidelity DNA polymerase, and NGS. By comparing the sequence of each read with the reference sequence, mutations were identified. The mutation can be identified to be due to an error introduced by either cDNA synthesis, PCR, or NGS based on whether the sequence reads with the same tag contain the same mutation or not. The error rates in cDNA synthesis with Moloney murine leukemia virus (MMLV) RT thermostable variant MM4 or the recently developed 16-tuple variant of family B DNA polymerase with RT activity, RTX, from Thermococcus kodakarensis, were 0.75-1.0 × 10-4 errors/base, while that in the reaction with the wild-type human immunodeficiency virus type 1 (HIV-1) RT was 2.6 × 10-4 errors/base. Overall, our method could precisely evaluate the fidelity of various RTs with different reaction conditions in a high-throughput manner without the use of expensive optics and troublesome adaptor ligation.

The cleavage site preference of the porcine pepsin on the N-terminal α1 chain of bovine type I collagen: a focal analysis with mass spectrometry.

Bovine type I collagen consists of two α1 and one α2 chains, containing the internal triple helical regions and the N- and C-terminal telopeptides. In industries, it is frequently digested with porcine pepsin to produce a triple helical collagen without the telopeptides. However, the digestion mechanism is not precisely understood. Here, we performed a mass spectrometric analysis of the pepsin digest of the N-terminal telopeptide pQLSYGYDEKSTGISVP (1-16) in the α1 chain. When purified collagen was digested, pQLSYGY (1-6) and pQLSYGYDEKSTG (1-12) were identified, while DEKSTG (7-12) was not. When the N-terminal telopeptide mimetic synthetic peptide pQLSK(MOCAc)GYDEKSTGISK(Dnp)P-NH2 was digested, pQLSK(MOCAc)GYDEKSTG (1-12) and ISK(Dnp)P-NH2 (13-16) were readily identified, pQLSK(MOCAc)GY (1-6) and DEKSTGISK(Dnp)P-NH2 (7-16) were weakly detected, and DEKSTG (7-12) was hardly identified. These results suggest that pepsin preferentially cleaves Tyr6-Asp7 and less preferentially Gly12-Ile13. They also suggest that the former cleavage requires native collagen structure, while the latter cleavage does not.

Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system.

We attempted to increase the thermostability of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). The eight-site saturation mutagenesis libraries corresponding to Ala70-Arg469 in the whole MMLV RT (Thr24-Leu671), in each of which 1 out of 50 amino acid residues was replaced with other amino acid residue, were constructed. Seven-hundred and sixty eight MMLV RT clones were expressed using a cell-free protein expression system, and their thermostabilities were assessed by the temperature of thermal treatment at which they retained cDNA synthesis activity. One clone D200C was selected as the most thermostable variant. The highest temperature of thermal treatment at which D200C exhibited cDNA synthesis activity was 57ºC, which was higher than for WT (53ºC). Our results suggest that a combination of site saturation mutagenesis library and cell-free protein expression system might be useful for generation of thermostable MMLV RT in a short period of time for expression and selection.

[A Case of Phrenic Nerve Paralysis During Adjuvant Chemotherapy for Rectal Cancer].

A man aged 66 years presented with pneumaturia as a major complaint. Cancer of the sigmoid colon with infiltration to the urinary bladder was diagnosed and the patient underwent colectomy of the sigmoid colon and partial cystectomy of the bladder in May 2015. Histopathologic examinations revealed pT4b, Si(bladder), pN(-), cM0, fStage II . Because intestinal sub-obstruction and lymphatic invasion were present, CapeOX was administered as an adjunctive chemotherapy for the high-risk Stage II cancer. Because Grade 2 peripheral neuropathy appeared as a side effect, the dose was decreased to 80% from the 3 cycle. After the 7 cycle, cough and disturbed breathing appeared. The chest CT scans did not reveal drug-induced interstitial pneumonia, but indicated an elevated right diaphragm and zosteroid changes in the medial lobe of the right lung due to discoid atelectatic condition. The Grade 1 respiratory symptoms were mild, and the lung field was considered to exhibit no problems. Thus, the 8 cycle was administered. The symptoms disappeared after about 2 weeks following completion of oral administration of capecitabine. The diaphragm also recovered to its original height. In the attached document, the frequency is unknown and "dyspnea" is written for L-OHP and capecitabine, respectively. It is unknown whether phrenic nerve paralysis occurs. However, because other organic lesions were absent and the symptoms appeared during chemotherapy, the possibility is not deniable. At present, 2 years postoperatively, recurrent lesions in the mediastinum and recurrent respiratory difficulties are absent. Generally, although phrenic nerve paralysis is not considered to be a specific side effect, it was considered that for respiratory difficulties, CT reveals not only the affected condition in the lung fields, but is also useful for detection.

[A Case of Unresectable Advanced Rectal Cancer with a Pancreatic Tumor That Was Successfully Treated with FOLFIRINOX].

A 72-year-old man was admitted to our hospital department in September 2014 because of a positive fecal occult blood test.Colonoscopy showed a type 2 tumor in half of the AV 15 cm rectosigmoid colon.Histology of the biopsy indicated a moderately differentiated adenocarcinoma, and the RAS gene test found wild type.On CT examination, there were multiple liver lung metastases and a 30mm diameter tumor with pancreatic duct extension to the pancreatic body.A PET-CT examination had a high SUVmax at the same site.Because of the location of the tumor EUS-FNA was not used.However, the possibility of pancreatic body cancer could not be denied after the CT examination.Treatment by radical resection was impossible because of the spread of the cancer so we selected chemotherapy.Undeniable pancreatic metastasis of rectal cancer, pancreatic cancer was used as a prognostic factor as double cancer of rectal cancer and pancreatic cancer, from that UGT1A1 test side effects appearance was a low-risk decision, was selected FOLFIRINOX in the treatment regimen.After 25 cycles, the pancreatic body tumor and liver metastases and also the primary tumor were reduced, the multiple lung metastases disappeared, and disease control was good.Side effects were diarrhea on the day of administration of irinotecan, but this was controllable by administering oral loperamide when starting the infusion.Grade 3 or more peripheral neuropathy has not developed, and this regimen is continuing.Pancreatic cancer is a solid cancer with a poor prognosis; if you do not reach the tissue diagnosis of metastatic pancreatic cancer, was a case in which no choice but to select a regimen to carcinoma of the prognostic.