The Japanese version of the Fear of COVID-19 scale: Reliability, validity, and relation to coping behavior.

COVID-19 is spreading worldwide, causing various social problems. The aim of the present study was to verify the reliability and validity of the Japanese version of the Fear of COVID-19 Scale (FCV-19S) and to ascertain FCV-19S effects on assessment of Japanese people's coping behavior. After back-translation of the scale, 450 Japanese participants were recruited from a crowdsourcing platform. These participants responded to the Japanese FCV-19S, the Japanese versions of the Hospital Anxiety and Depression scale (HADS) and the Japanese versions of the Perceived Vulnerability to Disease (PVD), which assesses coping behaviors such as stockpiling and health monitoring, reasons for coping behaviors, and socio-demographic variables. Results indicated the factor structure of the Japanese FCV-19S as including seven items and one factor that were equivalent to those of the original FCV-19S. The scale showed adequate internal reliability (α = .87; ω = .92) and concurrent validity, as indicated by significantly positive correlations with the Hospital Anxiety and Depression Scale (HADS; anxiety, r = .56; depression, r = .29) and Perceived Vulnerability to Disease (PVD; perceived infectability, r = .32; germ aversion, r = .29). Additionally, the FCV-19S not only directly increased all coping behaviors (ß = .21 - .36); it also indirectly increased stockpiling through conformity reason (indirect effect, ß = .04; total effect, ß = .31). These results suggest that the Japanese FCV-19S psychometric scale has equal reliability and validity to those of the original FCV-19S. These findings will contribute further to the investigation of various difficulties arising from fear about COVID-19 in Japan.

Quantitative measurement of a candidate serum biomarker peptide derived from α2-HS-glycoprotein, and a preliminary trial of multidimensional peptide analysis in females with pregnancy-induced hypertension.

Purpose We previously attempted to develop quantitative enzyme-linked immunosorbent assay (ELISA) systems for the PDA039/044/071 peptides, potential serum disease biomarkers (DBMs) of pregnancy-induced hypertension (PIH), primarily identified by a peptidomic approach (BLOTCHIP®-mass spectrometry (MS)). However, our methodology did not extend to PDA071 (cysteinyl α2-HS-glycoprotein341-367), due to difficulty to produce a specific antibody against the peptide. The aim of the present study was to establish an alternative PDA071 quantitation system using liquid chromatography-multiple reaction monitoring (LC-MRM)/MS, to explore the potential utility of PDA071 as a DBM for PIH. Methods We tested heat/acid denaturation methods in efforts to purify serum PDA071 and developed an LC-MRM/MS method allowing for specific quantitation thereof. We measured serum PDA071 concentrations, and these results were validated including by three-dimensional (3D) plotting against PDA039 (kininogen-1439-456)/044 (kininogen-1438-456) concentrations, followed by discriminant analysis. Results PDA071 was successfully extracted from serum using a heat denaturation method. Optimum conditions for quantitation via LC-MRM/MS were developed; the assayed serum PDA071 correlated well with the BLOTCHIP® assay values. Although the PDA071 alone did not significantly differ between patients and controls, 3D plotting of PDA039/044/071 peptide concentrations and construction of a Jackknife classification matrix were satisfactory in terms of PIH diagnostic precision. Conclusions Combination analysis using both PDA071 and PDA039/044 concentrations allowed PIH diagnostic accuracy to be attained, and our method will be valuable in future pathophysiological studies of hypertensive disorders of pregnancy.

Identification of an anti-sperm auto-monoclonal antibody (Ts4)-recognized molecule in the mouse sperm acrosomal region and its inhibitory effect on fertilization in vitro.

We previously established an anti-mouse sperm auto-monoclonal antibody, Ts4, which shows immunoreactivity against several kinds of glycoproteins in the acrosomal region of epididymal spermatozoa, testicular germ cells, and early embryo, via binding to an epitope containing a common N-linked oligosaccharide (OS) chain on the molecules. In mice, we have already demonstrated that the OS chain in the epitope for Ts4 is a fucosylated agalacto-complex-type biantennary glycan carrying bisecting N-acetylglucosamine. In the testis, one of the specific OS chain-conjugated molecules is TEX101, a germ cell-marker glycoprotein, which is expressed in spermatocytes, spermatids, and testicular spermatozoa, but not in epididymal spermatozoa. In this study, we identified a Ts4-reactive glycoprotein in mouse cauda epididymal sperm. An immunoprecipitation method together with liquid chromatography-tandem mass spectrometry showed that alpha-N-acetylglucosaminidase (Naglu; a degradation enzyme of heparan sulfate) is one of the glycoproteins recognized by Ts4 in the epididymal spermatozoa. Western blot and immunohistochemical analyses revealed that mouse Naglu exists in two forms (82 and 77kDa) and is expressed in the acrosomal region and the flagellum of cauda epididymal sperm. Of the two Naglu-forms expressed in sperm, Ts4 immunoreacted against only the 82-kDa form located on the acrosomal region. The Ts4 mAb and anti-Naglu pAb negatively affected mouse fertilization in vitro. In addition, Ts4 inhibited sperm acrosome reaction induced by heparan sulfate. The Ts4-recognized fucosylated agalactobiantennary complex-type glycan with bisecting N-acetylglucosamine and Naglu on cauda epididymal spermatozoa may play a role in the process of fertilization.

TEX101, a glycoprotein essential for sperm fertility, is required for stable expression of Ly6k on testicular germ cells.

TEX101, a germ cell-specific glycosyl-phosphatidylinositol (GPI)-anchored glycoprotein, is associated with Ly6k during spermatogenesis in testis. Although both Tex101(-/-) and Ly6k(-/-) mice can produce morphologically intact spermatozoa, both knockout mice show an infertile phenotype due to a disorder of spermatozoa to migrate into the oviduct. Since Ly6k specifically interacts with TEX101, complex formation of TEX101/Ly6k appears to be potentially important for functional sperm production. This study evaluated the fate of Ly6k in the presence or absence of TEX101 to explore the molecular interaction of both GPI-anchored proteins in seminiferous tubules. The present study showed that: 1) Although Ly6k mRNA was detected, the protein was present at very low levels in mature testes of Tex101(-/-) mice, 2) Ly6k mRNA level was within the normal range in Tex101(-/-) mice, 3) Ly6k mRNA was translated into a polypeptide in the testes of Tex101(+/+) and Tex101(-/-) mice, and 4) TEX101, as well as Ly6k, are co-factors that affect to molecular expression. These results indicate that both TEX101 and Ly6k contribute to the post-translational counterpart protein expression at the cell membrane. This mechanism may be important in maintaining the production of fertile spermatozoa during spermatogenesis.

Spatiotemporal expression of TRPM4 in the mouse cochlea.

The present study was conducted to elucidate the presence of the transient receptor potential cation channel subfamily M member 4, TRPM4, in the mouse inner ear. TRPM4 immunoreactivity (IR) was found in the cell body of inner hair cells (IHCs) in the organ of Corti in the apical side of marginal cells of the stria vascularis, in the apical portion of the dark cells of the vestibule, and in a subset of the type II neurons in the spiral ganglion. Subsequently, changes in the distribution and expression of TRPM4 in the inner ear during embryonic and postnatal developments were also evaluated. Immunohistochemical localization demonstrated that the emergence of the TRPM4-IR in IHCs occurs shortly before the onset of hearing, whereas that in the marginal cells happens earlier, at the time of birth, coinciding with the onset of endolymph formation. Furthermore, semiquantitative real-time PCR assay showed that expressions of TRPM4 in the organ of Corti and in the stria vascularis increased dramatically at the onset of hearing. Because TRPM4 is a Ca(2+) -activated monovalent-selective cation channel, these findings imply that TRPM4 contributes to potassium ion transport, essential for the signal transduction in IHCs and the formation of endolymph by marginal cells.

Epithelial Na⁺ sodium channels in magnocellular cells of the rat supraoptic and paraventricular nuclei.

The epithelial Na⁺ channels (ENaCs) are present in kidney and contribute to Na⁺ and water homeostasis. All three ENaC subunits (α, ß, and γ) were demonstrated in the cardiovascular regulatory centers of the rat brain, including the magnocellular neurons (MNCs) in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN). However, the functional significance of ENaCs in vasopressin (VP) and oxytocin (OT) synthesizing MNCs is completely unknown. In this study, we show with immunocytochemical double-labeling that the α-ENaC is colocalized with either VP or OT in MNCs in the SON and PVN. In addition, parvocellular neurons in the dorsal, ventrolateral, and posterior subregions of the PVN (not immunoreactive to VP or OT) are also immunoreactive for α-ENaC. In contrast, immunoreactivity to ß- and γ-ENaC is colocalized with VP alone within the MNCs. Furthermore, immunoreactivity for a known target for ENaC expression, the mineralcorticoid receptor (MR), is colocalized with both VP and OT in MNCs. Using single-cell RT-PCR, we detected mRNA for all three ENaC subunits and MR in cDNA libraries derived from single MNCs. In whole cell voltage clamp recordings, application of the ENaC blocker benzamil reversibly reduced a steady-state inward current and decreased cell membrane conductance approximately twofold. Finally, benzamil caused membrane hyperpolarization in a majority of VP and about one-half of OT neurons in both spontaneously firing and quiet cells. These results strongly suggest the presence of functional ENaCs that may affect the firing patterns of MNCs, which ultimately control the secretion of VP and OT.

Dependence of DNA double strand break repair pathways on cell cycle phase in human lymphoblastoid cells.

DNA double-strand breaks (DSBs) are usually repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR). NHEJ is thought to be the predominant pathway operating in mammalian cells functioning in all phases of the cell cycle, while HR works in the late-S and G2 phases. However, relative contribution, competition, and dependence on cell cycle phases are not fully understood. We previously developed a system to trace the fate of DSBs in the human genome by introducing the homing endonuclease I-SceI site into the thymidine kinase (TK) gene of human lymphoblastoid TK6 cells. Here, we use this system to investigate the relative contribution of HR and NHEJ for repairing I-SceI-induced DSBs under various conditions. We used a novel transfection system, Amaxa nucleofector, which directly introduces the I-SceI expression vector into cell nuclei. Approximately 65% of transfected cells expressed the I-SceI enzyme and over 50% of the cells produced a single DSB in the genome. The relative contribution of NHEJ and HR for repairing the DSB was approximately 100:1 and did not change with transfection efficiency. Cotransfection with KU80-siRNA significantly diminished KU80 protein levels and decreased NHEJ activity, but did not increase HR. We also investigated HR and NHEJ in synchronized cells. The HR frequency was 2-3 times higher in late-S/G2 phases than in G1, whereas NHEJ was unaffected. Even in late-S/G2 phases, NHEJ remained elevated relative to HR. Therefore, NHEJ is the major pathway for repairing endonuclease-induced DSBs in mammalian cells even in late-S/G2 phase, and does not compete with HR.

Potassium bromate treatment predominantly causes large deletions, but not GC>TA transversion in human cells.

Potassium bromate (KBrO(3)) is strongly carcinogenic in rodents and mutagenic in bacteria and mammalian cells in vitro. The proposed genotoxic mechanism for KBrO(3) is oxidative DNA damage. KBrO(3) can generate high yields of 8-hydroxydeoxyguanosine (8OHdG) DNA adducts, which cause GC>TA transversions in cell-free systems. In this study, we investigated the in vitro genotoxicity of KBrO(3) in human lymphoblastoid TK6 cells using the comet (COM) assay, the micronucleus (MN) test, and the thymidine kinase (TK) gene mutation assay. After a 4h treatment, the alkaline and neutral COM assay demonstrated that KBrO(3) directly yielded DNA damages including DNA double strand breaks (DSBs). KBrO(3) also induced MN and TK mutations concentration-dependently. At the highest concentration (5mM), KBrO(3) induced MN and TK mutation frequencies that were over 30 times the background level. Molecular analysis revealed that 90% of the induced mutations were large deletions that involved loss of heterozygosity (LOH) at the TK locus. Ionizing-irradiation exhibited similar mutational spectrum in our system. These results indicate that the major genotoxicity of KBrO(3) may be due to DSBs that lead to large deletions rather than to 8OHdG adducts that lead to GC>TA transversions, as is commonly believed. To better understand the genotoxic mechanism of KBrO(3), we analyzed gene expression profiles of TK6 cells using Affymetrix Genechip. Some genes involved in stress, apoptosis, and DNA repair were up-regulated by the treatment of KBrO(3). However, we could not observe the similarity of gene expression profile in the treatment of KBrO(3) to ionizing-irradiation as well as oxidative damage inducers.

Non-homologous end-joining for repairing I-SceI-induced DNA double strand breaks in human cells.

DNA double strand breaks (DSBs) are usually repaired through either non-homologous end-joining (NHEJ) or homologous recombination (HR). While HR is basically error-free repair, NHEJ is a mutagenic pathway that leads to deletion. NHEJ must be precisely regulated to maintain genomic integrity. To clarify the role of NHEJ, we investigated the genetic consequences of NHEJ repair of DSBs in human cells. Human lymphoblastoid cell lines TSCE5 and TSCE105 have, respectively, single and double I-SceI endonuclease sites in the endogenous thymidine kinase gene (TK) located on chromosome 17q. I-SceI expression generated DSBs at the TK gene. We used the novel transfection system (Amaxa Nucleofector) to introduce an I-SceI expression vector into the cells and randomly isolated clones. We found mutations involved in the DSBs in the TK gene in 3% of TSCE5 cells and 30% of TSCE105 cell clones. Most of the mutations in TSCE5 were small (1-30bp) deletions with a 0-4bp microhomology at the junction. The others consisted of large (>60) bp deletions, an insertion, and a rearrangement. Mutants resulting from interallelic HR also occurred, but infrequently. Most of the mutations in TSCE105, on the other hand, were deletions that encompassed the two I-SceI sites generated by NHEJ at DSBs. The sequence joint was similar to that found in TSCE5 mutants. Interestingly, some mutants formed a new I-SceI site by perfectly joining the two original I-SceI sites without deletion of the broken-ends. These results support the idea that NHEJ for repairing I-SceI-induced DSBs mainly results in small or no deletions. Thus, NHEJ must help maintain genomic integrity in mammalian cells by repairing DSBs as well as by preventing many deleterious alterations.

Genotoxicity of acrylamide and glycidamide in human lymphoblastoid TK6 cells.

The recent finding that acrylamide (AA), a potent carcinogen, is formed in foods during cooking raises human health concerns. In the present study, we investigated the genotoxicity of AA and its metabolite glycidamide (GA) in human lymphoblastoid TK6 cells examining three endpoints: DNA damage (comet assay), clastogenesis (micronucleus test) and gene mutation (thymidine kinase (TK) assay). In a 4 h treatment without metabolic activation, AA was mildly genotoxic in the micronucleus and TK assays at high concentrations (> 10 mM), whereas GA was significantly and concentration-dependently genotoxic at all endpoints at > or = 0.5 mM. Molecular analysis of the TK mutants revealed that AA predominantly induced loss of heterozygosity (LOH) mutation like spontaneous one while GA-induced primarily point mutations. These results indicate that the genotoxic characteristics of AA and GA were distinctly different: AA was clastogenic and GA was mutagenic. The cytotoxicity and genotoxicity of AA were not enhanced by metabolic activation (rat liver S9), implying that the rat liver S9 did not activate AA. We discuss the in vitro and in vivo genotoxicity of AA and GA.

Genotoxicity of microcystin-LR in human lymphoblastoid TK6 cells.

Toxic cyanobacteria (blue-green algae) water blooms have become a serious problem in several industrialized areas of the world. Microcystin-LR (MCLR) is a cyclic heptapeptidic toxin produced by the cyanobacteria. In the present study, we used human lymphoblastoid cell line TK6 to investigate the in vitro genotoxicity of MCLR. In a standard 4h treatment, MCLR did not induce a significant cytotoxic response at <80 microg/ml. In a prolonged 24h treatment, in contrast, it induced cytotoxic as well as mutagenic responses concentration-dependently starting at 20 microg/ml. At the maximum concentration (80 microg/ml), the micronucleus frequency and the mutation frequency at the heterozygous thymidine kinase (TK) locus were approximately five-times the control values. Molecular analysis of the TK mutants revealed that MCLR specifically induced loss of heterozygosity at the TK locus, but not point mutations or other small structural changes. These results indicate that MCLR had a clastogenic effect. We discuss the mechanisms of MCLR genotoxicity and the possibility of its being a hepatocarcinogen.

Deletion, rearrangement, and gene conversion; genetic consequences of chromosomal double-strand breaks in human cells.

Chromosomal double-strand breaks (DSBs) in mammalian cells are usually repaired through either of two pathways: end-joining (EJ) or homologous recombination (HR). To clarify the relative contribution of each pathway and the ensuing genetic changes, we developed a system to trace the fate of DSBs that occur in an endogenous single-copy human gene. Lymphoblastoid cell lines TSCE5 and TSCER2 are heterozygous (+/-) or compound heterozygous (-/-), respectively, for the thymidine kinase gene (TK), and we introduced an I-SceI endonuclease site into the gene. EJ for a DSB at the I-SceI site results in TK-deficient mutants in TSCE5 cells, while HR between the alleles produces TK-proficient revertants in TSCER2 cells. We found that almost all DSBs were repaired by EJ and that HR rarely contributes to the repair in this system. EJ contributed to the repair of DSBs 270 times more frequently than HR. Molecular analysis of the TK gene showed that EJ mainly causes small deletions limited to the TK gene. Seventy percent of the small deletion mutants analyzed showed 100- to 4,000-bp deletions with a 0- to 6-bp homology at the joint. Another 30%, however, were accompanied by complicated DNA rearrangements, presumably the result of sister-chromatid fusion. HR, on the other hand, always resulted in non-crossing-over gene conversion without any loss of genetic information. Thus, although HR is important to the maintenance of genomic stability in DNA containing DSBs, almost all chromosomal DSBs in human cells are repaired by EJ.