Diminished nuclear RNA decay upon Salmonella infection upregulates antibacterial noncoding RNAs.

Cytoplasmic mRNA degradation controls gene expression to help eliminate pathogens during infection. However, it has remained unclear whether such regulation also extends to nuclear RNA decay. Here, we show that 145 unstable nuclear RNAs, including enhancer RNAs (eRNAs) and long noncoding RNAs (lncRNAs) such as NEAT1v2, are stabilized upon Salmonella infection in HeLa cells. In uninfected cells, the RNA exosome, aided by the Nuclear EXosome Targeting (NEXT) complex, degrades these labile transcripts. Upon infection, the levels of the exosome/NEXT components, RRP6 and MTR4, dramatically decrease, resulting in transcript stabilization. Depletion of lncRNAs, NEAT1v2, or eRNA07573 in HeLa cells triggers increased susceptibility to Salmonella infection concomitant with the deregulated expression of a distinct class of immunity-related genes, indicating that the accumulation of unstable nuclear RNAs contributes to antibacterial defense. Our results highlight a fundamental role for regulated degradation of nuclear RNA in the response to pathogenic infection.

Differential oxidation processes of peroxiredoxin 2 dependent on the reaction with several peroxides in human red blood cells.

Peroxiredoxins (Prxs) detoxify hydrogen peroxide (H2O2), peroxynitrite, and various organic hydroperoxides. However, the differential oxidative status of Prxs reacted with each peroxide remains unclear. In the present study, we focused on the oxidative alteration of Prxs and demonstrated that, in human red blood cells (RBCs), peroxiredoxin 2 (Prx2) is readily reactive with H2O2, forming disulfide dimers, but was not easily hyperoxidized. In contrast, Prx2 was highly sensitive to the relatively hydrophobic oxidants, such as tert-butyl hydroperoxide (t-BHP) and cumene hydroperoxide. These peroxides hyperoxidized Prx2 into oxidatively damaged forms in RBCs. The t-BHP treatment formed hyperoxidized Prx2 in a dose-dependent manner. When organic hydroperoxide-treated RBC lysates were subjected to reverse-phase high performance liquid chromatography, two peaks derived from hyperoxidized Prx2 appeared along with the decrease of that corresponding to native Prx2. Liquid chromatography-tandem mass spectrometry analysis clearly showed that hyperoxidation to sulfonic acid (-SO3H) at Cys-51 residue was more advanced in a newfound hyperoxidized Prx2 compared to another hydrophobic hyperoxidized form previously identified. These results indicate that irreversible hyperoxidation of the Prx2 monomer in RBCs was easily caused by organic hydroperoxide but not H2O2. Thus, it is important to detect the hyperoxidation of Prx2 into sulfinic or sulfonic acid derivates of Cys-51 because hyperoxidized Prx2 is a potential marker of oxidative injury caused by organic hydroperoxides in human RBCs.

WDR74 participates in an early cleavage of the pre-rRNA processing pathway in cooperation with the nucleolar AAA-ATPase NVL2.

WD repeat-containing protein 74 (WDR74), a nucleolar-localized protein, is the mammalian ortholog of Nsa1, a 60S ribosome assembly factor in yeast. We previously showed that WDR74 associates with MTR4, the nuclear exosome-assisting RNA helicase, whose dissociation is prohibited by an ATPase-deficient mutant of the AAA-type chaperone NVL2. However, the functions and regulation of WDR74 during ribosome biogenesis in cooperation with NVL2 remains unknown. Here, we demonstrated that knockdown of WDR74 leads to significant defects in the pre-rRNA cleavage within the internal transcribed spacer 1 (ITS1), occurring in an early stage of the processing pathway. Interestingly, when the dissociation of WDR74 from the MTR4-containing exonuclease complex was impaired upon expression of the mutant NVL2, the same processing defect, with partial migration of WDR74 from the nucleolus towards the nucleoplasm, was observed. In the nucleoplasm, an increased interaction between WDR74 and MTR4 was detected by in situ proximity ligation assay. Therefore, the dissociation of WDR74 from MTR4 in a late stage of rRNA synthesis is thought to be required for appropriate maturation of the pre-60S particles. These results suggest that the spatiotemporal regulation of ribosome biogenesis in the nucleolus is mediated by the ATPase activity of NVL2.

[Effect of Japanese Traditional Medicine, Ninjin-Youei-To(TJ-108), on the Quality of Life of Patients with Non-Small Cell Lung Cancer Receiving Outpatient Chemotherapy].

An increasing number of patients with lung cancer are undergoing outpatient chemotherapy, and thus, it is very important to maintain the quality of life(QOL)of these patients. Ninjin-Youei-To(TJ-108), a Japanese traditional medicine, has been reported to improve the QOL of patients with advanced cancer. However, the effect of TJ-108 in patients with lung cancer undergoing outpatient chemotherapy is unknown. Therefore, we conducted this study. To investigate factors influencing the QOL of these patients, we administered a QOL questionnaire,"The QOL Questionnaire for Cancer Patients Treated with Anticancer Drugs"(QOL-ACD)to 15 patients with non-small cell lung cancer. Factors related to the overall QOL scores and other categories indicating"activity","physical condition","psychological condition","social relationship", and"face scale" were analyzed. No significant decrease in each of the evaluated factors was observed in this study.

AAA-ATPase NVL2 acts on MTR4-exosome complex to dissociate the nucleolar protein WDR74.

Nuclear VCP-like 2 (NVL2) is a chaperone-like nucleolar ATPase of the AAA (ATPase associated with diverse cellular activities) family, which exhibits a high level of amino acid sequence similarity with the cytosolic AAA-ATPase VCP/p97. These proteins generally act on macromolecular complexes to stimulate energy-dependent release of their constituents. We previously showed that NVL2 interacts with RNA processing/degradation machinery containing an RNA helicase MTR4/DOB1 and an exonuclease complex, nuclear exosome, and involved in the biogenesis of 60S ribosomal subunits. These observations implicate NVL2 as a remodeling factor for the MTR4-exosome complex during the maturation of pre-ribosomal particles. Here, we used a proteomic screen and identified a WD repeat-containing protein 74 (WDR74) as a factor that specifically dissociates from this complex depending on the ATPase activity of NVL2. WDR74 shows weak amino acid sequence similarity with the yeast ribosome biogenesis protein Nsa1 and is co-localized with NVL2 in the nucleolus. Knockdown of WDR74 decreases 60S ribosome levels. Taken together, our results suggest that WDR74 is a novel regulatory protein of the MTR4-exsosome complex whose interaction is regulated by NVL2 and is involved in ribosome biogenesis.

NVL2, a nucleolar AAA-ATPase, is associated with the nuclear exosome and is involved in pre-rRNA processing.

Nuclear VCP-like 2 (NVL2) is a member of the chaperone-like AAA-ATPase family and is involved in the biosynthesis of 60S ribosomal subunits in mammalian cells. We previously showed the interaction of NVL2 with a DExD/H-box RNA helicase MTR4/DOB1, which is a known cofactor for an exoribonuclease complex, the exosome. This finding implicated NVL2 in RNA metabolic processes during ribosome biogenesis. In the present study, we found that a series of mutations within the ATPase domain of NVL2 causes a defect in pre-rRNA processing into mature 28S and 5.8S rRNAs. Co-immunoprecipitation analysis showed that NVL2 was associated with the nuclear exosome complex, which includes RRP6 as a nucleus-specific catalytic subunit. This interaction was prevented by depleting either MTR4 or RRP6, indicating their essential role in mediating this interaction with NVL2. Additionally, knockdown of MPP6, another cofactor for the nuclear exosome, also prevented the interaction by causing MTR4 to dissociate from the nuclear exosome. These results suggest that NVL2 is involved in pre-rRNA processing by associating with the nuclear exosome complex and that MPP6 is required for maintaining the integrity of this rRNA processing complex.

Elemental Distribution in Multilayer Systems by Laser-Assisted Atom Probe Tomography with Various Analysis Directions.

Elemental distributions in a magnetic multilayer system with the structure Si substrate/Ta/NiFe/Ru/CoFeB/Ru/NiFe were studied using atom probe tomography (APT) along different analysis directions. The distributions of Ru and B atoms, which require a high evaporation field, were strongly influenced by the APT analysis direction. In particular, B in the CoFeB layer appeared near the interface with the lower Ru layer when the analysis was anti-parallel to the film growth direction, while B atoms were observed at the other side of the CoFeB layer when the analysis was parallel to the film growth direction. Moreover, when the analysis was perpendicular to the film growth direction, a homogenous distribution of B atoms was found within the CoFeB layer. Owing to this B behavior, the underlying Ru layer was affected in both of these analysis directions. In APT measurements of such a multilayer system composed of a stack of different evaporation field materials, evaluation of the elemental distribution around interfaces should be performed from more than one analysis direction.

Human exosomal placenta-associated miR-517a-3p modulates the expression of PRKG1 mRNA in Jurkat cells.

During pregnancy, human placenta-associated microRNAs (miRNAs) derived from the miRNA cluster in human chromosome 19 are expressed in villous trophoblasts and secreted into maternal circulation via exosomes; however, little is known about whether circulating placenta-associated miRNAs are transferred into maternal immune cells via exosomes, and modulate expression of target genes in the recipient cells. We employed an in vitro model of trophoblast-immune cell communication using BeWo cells (a human trophoblast cell line) and Jurkat cells (a human leukemic T-cell line) and investigated whether BeWo exosomal placenta-associated miRNAs can suppress expression of target genes in the recipient Jurkat cells. Using this system, we identified PRKG1 as a target gene of placenta-associated miRNA miR-517a-3p. Moreover, we demonstrated that BeWo exosomal miR-517a-3p was internalized into Jurkat cells and subsequently suppressed the expression of PRKG1 in recipient Jurkat cells. Furthermore, using peripheral blood natural killer (NK) cells in vivo, we confirmed that circulating miR-517a-3p was delivered into maternal NK cells as it was into Jurkat cells in vitro. Placenta-associated miR-517a-3p was incorporated into maternal NK cells in the third trimester, and it was rapidly cleared after delivery. Expression levels of miR-517a-3p and its target mRNA PRKG1 were inversely correlated in NK cells before and after delivery. These in vitro and in vivo results suggest that exosome-mediated transfer of placenta-associated miRNAs and subsequent modulation of their target genes occur in maternal NK cells. The present study provides novel insight into our understanding of placenta-maternal communication.

Proteomic analysis of adult T-cell leukemia/lymphoma: A biomarker identification strategy based on preparation and in-solution digestion methods of total proteins.

Adult T-cell leukemia/lymphoma (ATL), caused by human T-cell leukemia virus type-1 (HTLV-1) infection, is a malignant hematologic cancer that remains difficult to cure. We herein established a biomarker identification strategy based on the total cell proteomics of cultured ATL cells to search for novel ATL biomarkers. Four protocols with a combination of selected conditions based on lysis buffers and addition agents for total cell proteomics were used for a differential analysis between the ATL cell group (consisting of 11 cell lines), HTLV-1-infected cell group (consisting of 6 cell lines), and HTLV-1-negative cell group (consisting of 6 cell lines). In the analysis, we identified 24 and 27 proteins that were significantly increased (ratio ≥2.0, p < 0.05) and decreased (ratio ≤ 0.5, p < 0.05), respectively, in the ATL group. Previously reported CCL3 and CD30/TNFRSF8 were confirmed to be among significantly increased proteins. Furthermore, correlation analysis between identified proteins and Tax suggested that RASSF2 and GORASP2 were candidates of novel Tax-regulated factors. The biomarker identification strategy established herein is expected to contribute to the identification of biomarkers for ATL and other diseases.

Nanoscale magnetic characterization of tunneling magnetoresistance spin valve head by electron holography.

Nanostructured magnetic materials play an important role in increasing miniaturized devices. For the studies of their magnetic properties and behaviors, nanoscale imaging of magnetic field is indispensible. Here, using electron holography, the magnetization distribution of a TMR spin valve head of commercial design is investigated without and with a magnetic field applied. Characterized is the magnetic flux distribution in complex hetero-nanostructures by averaging the phase images and separating their component magnetic vectors and electric potentials. The magnetic flux densities of the NiFe (shield and 5 nm-free layers) and the CoPt (20 nm-bias layer) are estimated to be 1.0 T and 0.9 T, respectively. The changes in the magnetization distribution of the shield, bias, and free layers are visualized in situ for an applied field of 14 kOe. This study demonstrates the promise of electron holography for characterizing the magnetic properties of hetero-interfaces, nanostructures, and catalysts.

Electron holography study of magnetization behavior in the writer pole of a perpendicular magnetic recording head by a 1 MV transmission electron microscope.

The magnetic domain structure of the writer poles of perpendicular magnetic recording heads was studied using electron holography. Although the domain structure of a 100-nm-thick writer pole could be observed with a 300 kV transmission electron microscope, that of the 250-nm-thick writer pole could not be analyzed due to the limited transmission capability of the instrument. On the other hand, the detailed domain structure of the 250-nm-thick writer pole was successfully analyzed by a 1 MV electron microscope using its high transmission capability. The thickness and material dependency of the domain structure of a writer pole were discussed.

Hyperoxidized Peroxiredoxin 2 Is a Possible Biomarker for the Diagnosis of Obstructive Sleep Apnea.

Peroxiredoxin (Prx) 2 in red blood cells (RBCs) reacts with various reactive oxygen species and changes to hyperoxidized Prx2 (Prx2-SO2/3). Therefore, Prx2 may serve as an indicator of oxidative stress in vivo. This study aimed to analyze Prx2-SO2/3 levels in clinical samples to examine whether the oxidation state of Prx2 in human RBCs reflects the pathological condition of oxidative stress diseases. We first focused on obstructive sleep apnea (OSA), a hypoxic stress-induced disease of the respiratory system, and investigated the levels of Prx2-SO2/3 accumulated in the RBCs of OSA patients. In measurements on a small number of OSA patients and healthy subjects, levels of Prx2-SO2/3 accumulation in patients with OSA were clearly increased compared to those in healthy subjects. Hence, we proceeded to validate these findings with more samples collected from patients with OSA. The results revealed significantly higher levels of erythrocytic Prx2-SO2/3 in patients with OSA than in healthy subjects, as well as a positive correlation between the severity of OSA and Prx2-SO2/3 levels in the RBCs. Moreover, we performed a chromatographic study to show the structural changes of Prx2 due to hyperoxidation. Our findings demonstrated that the Prx2-SO2/3 molecules in RBCs from patients with OSA were considerably more hydrophilic than the reduced form of Prx2. These results implicate Prx2-SO2/3 as a promising candidate biomarker for OSA.

Transposable elements and an epigenetic basis for punctuated equilibria.

Evolution is frequently concentrated in bursts of rapid morphological change and speciation followed by long-term stasis. We propose that this pattern of punctuated equilibria results from an evolutionary tug-of-war between host genomes and transposable elements (TEs) mediated through the epigenome. According to this hypothesis, epigenetic regulatory mechanisms (RNA interference, DNA methylation and histone modifications) maintain stasis by suppressing TE mobilization. However, physiological stress, induced by climate change or invasion of new habitats, disrupts epigenetic regulation and unleashes TEs. With their capacity to drive non-adaptive host evolution, mobilized TEs can restructure the genome and displace populations from adaptive peaks, thus providing an escape from stasis and generating genetic innovations required for rapid diversification. This "epi-transposon hypothesis" can not only explain macroevolutionary tempo and mode, but may also resolve other long-standing controversies, such as Wright's shifting balance theory, Mayr's peripheral isolates model, and McClintock's view of genome restructuring as an adaptive response to challenge.

Interactome analysis of the Tudor domain-containing protein SPF30 which associates with the MTR4-exosome RNA-decay machinery under the regulation of AAA-ATPase NVL2.

The AAA-ATPase NVL2 associates with an RNA helicase MTR4 and the nuclear RNA exosome in the course of ribosome biogenesis. In our proteomic screen, we had identified a ribosome biogenesis factor WDR74 as a MTR4-interacting partner, whose dissociation is stimulated by the ATP hydrolysis of NVL2. In this study, we report the identification of splicing factor 30 (SPF30), another MTR4-interacting protein with a similar regulatory mechanism. SPF30 is a pre-mRNA splicing factor harboring a Tudor domain in its central region, which regulates various cellular events by binding to dimethylarginine-modified proteins. The interaction between SPF30 and the exosome core is mediated by MTR4 and RRP6, a catalytic component of the nuclear exosome. The N- and C-terminal regions, but not the Tudor domain, of SPF30 are involved in the association with MTR4 and the exosome. The knockdown of SPF30 caused subtle delay in the 12S pre-rRNA processing to mature 5.8S rRNA, even though no obvious effect was observed on the ribosome subunit profile in the cells. Shotgun proteomic analysis to search for SPF30-interacting proteins indicated its role in ribosome biogenesis, pre-mRNA splicing, and box C/D snoRNA biogenesis. These results suggest that SPF30 collaborates with the MTR4-exosome machinery to play a functional role in multiple RNA metabolic pathways, some of which may be regulated by the ATP hydrolysis of NVL2.

Accumulation of Carbonyl Proteins in the Brain of Mouse Model for Methylglyoxal Detoxification Deficits.

Recent studies have shown that carbonyl stress is a causative factor of schizophrenia, categorized as carbonyl stress-related schizophrenia (CS-SCZ). However, the correlation between carbonyl stress and the pathogenesis of this disease is not well established. In this study, glyoxalase 1(Glo1)-knockout and vitamin B6-deficient mice (KO/VB6 (-) mice), which are susceptible to methylglyoxal (MGO)-induced oxidative damages, were used as a CS-SCZ model to analyze MGO-modified protein and the carbonyl stress status in the brain. A comparison between Wild/VB6(+) mice and KO/VB6(-) mice for accumulated carbonyl proteins levels, with several advanced glycation end products (AGEs) in the brain, revealed that carbonyl protein levels with the Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl) ornithine (MG-H1) moiety were significantly increased in the hippocampus, prefrontal cortex, striatum, cerebral cortex, and brainstem regions of the brain in KO/VB6(-) mice. Moreover, two-dimensional electrophoresis and Liquid chromatography-tandem mass spectrometry analysis showed MG-H1-modified arginine residues in mitochondrial creatine kinase, beta-adrenergic receptor kinase 1, and T-complex protein in the hippocampus region of KO/VB6(-) mice, but not in Wild/VB6(+) mice. In particular, MG-H1 modification of mitochondrial creatine kinase was quite notable. These results suggest that further studies focusing on MG-H1-modified and accumulated proteins in the hippocampus may reveal the onset mechanism of CS-SCZ induced by MGO-induced oxidative damages.

Correlation of the Commercial Anti-SARS-CoV-2 Receptor Binding Domain Antibody Test with the Chemiluminescent Reduction Neutralizing Test and Possible Detection of Antibodies to Emerging Variants.

Serological tests are beneficial for recognizing the immune response against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). To identify protective immunity, optimization of the chemiluminescent reduction neutralizing test (CRNT) is critical. Whether commercial antibody tests have comparable accuracy is unknown. Serum samples were obtained from COVID-19 patients (n = 74), SARS-CoV-2 PCR-negative (n = 179), and suspected healthy individuals (n = 229) before SARS-CoV-2 variants had been detected locally. The convalescent phase was defined as the period after day 10 from disease onset or the episode of close contact. The CRNT using pseudotyped viruses displaying the wild-type (WT) spike protein and a commercial anti-receptor-binding domain (RBD) antibody test were assayed. Serology for the B.1.1.7 and B.1.351 variants was also assayed. Both tests concurred for symptomatic COVID-19 patients in the convalescent phase. They clearly differentiated between patients and suspected healthy individuals (sensitivity: 95.8% and 100%, respectively; specificity: 99.1% and 100%, respectively). Anti-RBD antibody test results correlated with neutralizing titers (r = 0.31, 95% confidence interval [CI] 0.22-0.38). Compared with the WT, lower CRNT values were observed for the variants. Of the samples with ≥100 U/mL by the anti-RBD antibody test, 77.8% and 88.9% showed ≥50% neutralization against the B.1.1.7 and the B.1.351 variants, respectively. Exceeding 100 U/mL in the anti-RBD antibody test was associated with neutralization of variants (P < 0.01). The CRNT and commercial anti-RBD antibody test effectively classified convalescent COVID-19 patients. Strong positive results with the anti-RBD antibody test can reflect neutralizing activity against emerging variants. IMPORTANCE This study provides a diagnostic evidence of test validity, which can lead to vaccine efficacy and proof of recovery after COVID-19. It is not easy to know neutralization against SARS-CoV-2 in the clinical laboratory because of technical and biohazard issues. The correlation of the quantitative anti-receptor-binding domain antibody test, which is widely available, with neutralizing test indicates that we can know indirectly the state of acquisition of functional immunity against wild and variant-type viruses in the clinical laboratory.

Quantitative evaluation of magnetic flux density in a magnetic recording head and pseudo soft underlayer by electron holography.

The magnetic interaction between the pole tip of a single-pole head and a pseudo soft underlayer in perpendicular magnetic recording was observed by electron holography. The magnetic flux density inside the soft underlayer was quantitatively evaluated. The distribution of magnetic flux density was calculated using the finite element method, and the influences of the modulation of the reference wave and stray fields were investigated by comparison with experimental results. The flux density observed was found to be underestimated due to the modulation of the phase shift in reference wave. The magnetic flux measured experimentally was larger than that inside the specimen because of the relatively large stray fields above and below the specimen in the direction of the electron beam.

[Identification of Heparin-binding Proteins on the Cell Surface of Cryptococcus neoformans].

Interactions between virulence factors of pathogens and host responses play an important role in the establishment of infection by microbes. We focused on interactions between Cryptococcus neoformans proteins and heparin, which is abundant on host epithelial cells. Surface proteins were extracted and analyzed. Fractions from anion-exchange column chromatography interacted with heparin in surface plasmon resonance analyses. Heparin-binding proteins were purified and then separated by gel electrophoresis; and were identified as transaldolase, glutathione-disulfide reductase, and glyoxal oxidase. These results imply that multifunctional molecules on C. neoformans cells, such as those involved in heparin binding, may play roles in adhesion that trigger responses in the host.

Sewall Wright, shifting balance theory, and the hardening of the modern synthesis.

The period between the 1940s and 1960s saw the hardening of the modern synthesis in evolutionary biology. Gould and Provine argue that Wright's shifting balance theory of evolution hardened during this period. But their account does not do justice to Wright, who always regarded selection as acting together with drift. This paper presents a more adequate account of the development of Wright's shifting balance theory, paying particular attention to his application of the theory to the geographical distribution of flower color dimorphism in Linanthus parryae. The account shows that even in the heyday of the hardened synthesis, the balance or interaction of evolutionary factors, such as drift, selection, and migration, occupied pride of place in Wright's theory, and that between the 1940s and 1970s, Wright developed the theory of isolation by distance to quantitatively represent the structure of the Linanthus population, which he argued had the kind of structure posited by his shifting balance theory. In the end, Wright arrived at a sophisticated description of the structure of the Linanthus population, where the interaction between drift and selection varied spatially.

Peroxidatic cysteine residue of peroxiredoxin 2 separated from human red blood cells treated by tert-butyl hydroperoxide is hyperoxidized into sulfinic and sulfonic acids.

Peroxiredoxin 2 (Prx2) is a redox enzyme that is abundantly expressed in red blood cells (RBCs) and has been the focus of clinical attention for monitoring the oxidative status. We previously developed a method to quantify the reduced and hyperoxidized forms of Prx2 in human RBCs using reverse-phase high-performance liquid chromatography (HPLC). In the present study, we investigated the hyperoxidative status of Prx2 at the molecular level in a post-translational modification analysis using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. The LC-MS/MS analysis of the trypsin digests of Prx2 fractionated by reverse-phase HPLC demonstrated that the cysteine-51 residue (Cys-51) of the protein was modified with the hyperoxidative functional groups, sulfinic acid (-SO2H) and sulfonic acid (-SO3H), in RBCs treated with tert-butyl hydroperoxide (t-BHP). Furthermore, a selected ion monitoring (SIM) analysis quantitatively showed that sulfinic acid- and sulfonic acid-induced modifications in Prx2 Cys-51 were increased by the treatment with the oxidant. It was demonstrated that the peroxidatic cysteine of Prx2 separated using our HPLC system for oxidative monitoring was hyperoxidized into sulfinic acid and sulfonic acid in RBCs under an oxidative stress condition.