Effect of the COVID-19 pandemic lockdown on the physique of school-age children in Japan.

PURPOSE: Schools in Japan were closed nationwide from March to May 2020 because of the coronavirus disease 2019 (COVID-19) pandemic. Many suspect that this school closure affected children's mental and physical health. We investigated changes in school-age children's physiques to determine the effects of the COVID-19 lockdown and restrictions on their health. METHODS: Data were extracted from a database of school physical examinations in Osaka elementary and junior high schools for 4 consecutive years from 2018 to 2021. The following characteristics were analyzed: short stature, tall stature, underweight, mild obesity, middle grade obesity, and severe obesity. The paired Student t-test was used to compare school examination data in the prepandemic period (2018-2019), pandemic lockdown (2019-2020), and post-lockdown period (2020-2021). RESULTS: Obesity rates in elementary school students aged 6-12 years, particularly in boys, were significantly higher during the lockdown than they were in 2019. After the pandemic, the tall stature rate continued to rise, while rates of short stature and underweight decreased in both sexes in 2020. In junior high school students aged 12-15 years, rates of obesity and underweight tended to decrease in 2020. However, these rates rebounded and rose in 2021 when the lockdown was lifted. CONCLUSION: During the COVID-19 pandemic lockdown, elementary school students gained weight, while junior high school students lost weight. The lockdown that was implemented during the COVID-19 pandemic had an unfavorable effect on weight gain, particularly in young school-age children.

NEAT1-SOD2 Axis Confers Sorafenib and Lenvatinib Resistance by Activating AKT in Liver Cancer Cell Lines.

This study investigated the effects of a long noncoding RNA, nuclear paraspeckle assembly transcript 1 (NEAT1) variant 1 (NEAT1v1) on drug resistance in liver cancer cell lines. NEAT1 knockdown activated mitogen-activated protein kinase (MAPK) signaling pathways, including MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK), but suppressed AKT. Moreover, NEAT1 knockdown sensitized liver cancer cells to sorafenib and lenvatinib, both clinically used for treating hepatocellular carcinoma, whereas it conferred resistance to an AKT-targeted drug, capivasertib. NEAT1v1 overexpression suppressed MEK/ERK and activated AKT, resulting in resistance to sorafenib and lenvatinib and sensitization to capivasertib. Superoxide dismutase 2 (SOD2) knockdown reverted the effects of NEAT1v1 overexpression on the sensitivity to the molecular-targeted drugs. Although NEAT1 or SOD2 knockdown enhanced endoplasmic reticulum (ER) stress, concomitant with the suppression of AKT, taurodeoxycholate, an ER stress suppressor, did not restore AKT activity. Although further in vivo and clinical studies are needed, these results suggested that NEAT1v1 switches the growth modality of liver cancer cell lines from MEK/ERK-dependent to AKT-dependent mode via SOD2 and regulates sensitivity to the molecular-targeted drugs independent of ER stress.

NEAT1 Confers Radioresistance to Hepatocellular Carcinoma Cells by Inducing PINK1/Parkin-Mediated Mitophagy.

A long noncoding RNA, nuclear paraspeckle assembly transcript 1 (NEAT1) variant 1 (NEAT1v1), confers radioresistance to hepatocellular carcinoma (HCC) cells by inducing autophagy via γ-aminobutyric acid A receptor-associated protein (GABARAP). Radiation induces oxidative stress to damage cellular components and organelles, but it remains unclear how NEAT1v1 protects HCC cells from radiation-induced oxidative stress via autophagy. To address this, we precisely investigated NEAT1v1-induced autophagy in irradiated HCC cell lines. X-ray irradiation significantly increased cellular and mitochondrial oxidative stress and mitochondrial DNA content in HCC cells while NEAT1v1 suppressed them. NEAT1v1 concomitantly induced the phosphatase and tensin homolog-induced kinase 1 (PINK1)/parkin-mediated mitophagy. Interestingly, parkin expression was constitutively upregulated in NEAT1v1-overexpressing HCC cells, leading to increased mitochondrial parkin levels. Superoxide dismutase 2 (SOD2) was also upregulated by NEAT1v1, and GABARAP or SOD2 knockdown in NEAT1v1-overexpressing cells increased mitochondrial oxidative stress and mitochondrial DNA content after irradiation. Moreover, it was suggested that SOD2 was involved in NEAT1v1-induced parkin expression, and that GABARAP promoted parkin degradation via mitophagy. This study highlights the unprecedented roles of NEAT1v1 in connecting radioresistance and mitophagy in HCC.

Impact of childhood growth and obesity curves in school health examinations.

BACKGROUND: With the revision of the Japanese School Health and Safety Law in 2016, the use of growth and obesity curves has been recommended. This study aimed to determine the prevalence of growth and obesity curve creation in elementary and junior high schools using government-issued software in Japan between 2016 and 2019. METHODS: A questionnaire survey was conducted with school nursing teachers in elementary and junior high schools in Osaka, Japan. The questionnaire was distributed and collected by e-mail between 1 and 31 March 2020. RESULTS: The survey response rate was 87.1%. In total, 78.5% of the elementary schools, and 75.0% of the junior high schools had the software for creating the growth curves. The rate of adoption of growth curve creation using the software increased in elementary schools (from 16.2% in 2016 to 40.5% in 2019 and in junior high schools from 6.0% in 2016 to 33.6% in 2019. The detection rates of growth abnormalities also increased over the 4 years in elementary and junior high schools, as follows: short stature (2.48- and 3.81-fold, respectively), tall stature (2.77- and 4.77-fold, respectively), emaciation (2.62 and 4.85-fold, respectively), mild obesity (2.66 and 5.15-fold, respectively), moderate obesity (2.71- and 4.14-fold, respectively), and severe obesity (2.45- and 3.32-fold, respectively). The rates of receiving a recommendation slip and going on to consult a specialist for each growth abnormality were low. CONCLUSIONS: By utilizing these curves, the detection rate of physical development abnormalities increased, but the rate of recommending a specialist consultation and the rate of actual consultation with a specialist were still low.

NEAT1 Confers Radioresistance to Hepatocellular Carcinoma Cells by Inducing Autophagy through GABARAP.

A long noncoding RNA (lncRNA), nuclear enriched abundant transcript 1 (NEAT1) variant 1 (NEAT1v1), is involved in the maintenance of cancer stem cells (CSCs) in hepatocellular carcinoma (HCC). CSCs are suggested to play important roles in therapeutic resistance. Therefore, we investigated whether NEAT1v1 is involved in the sensitivity to radiation therapy in HCC. Gene knockdown was performed using short hairpin RNAs, and NEAT1v1-overexpressing HCC cell lines were generated by stable transfection with a NEAT1v1-expressing plasmid DNA. Cells were irradiated using an X-ray generator. We found that NEAT1 knockdown enhanced the radiosensitivity of HCC cell lines and concomitantly inhibited autophagy. NEAT1v1 overexpression enhanced autophagy in the irradiated cells and conferred radioresistance. Gamma-aminobutyric acid receptor-associated protein (GABARAP) expression was downregulated by NEAT1 knockdown, whereas it was upregulated in NEAT1v1-overexpressing cells. Moreover, GABARAP was required for NEAT1v1-induced autophagy and radioresistance as its knockdown significantly inhibited autophagy and sensitized the cells to radiation. Since GABARAP is a crucial protein for the autophagosome-lysosome fusion, our results suggest that NEAT1v1 confers radioresistance to HCC by promoting autophagy through GABARAP.

NEAT1 is Required for the Expression of the Liver Cancer Stem Cell Marker CD44.

CD44, a cancer stem cell (CSC) marker, is required for maintaining CSC properties in hepatocellular carcinoma (HCC). Nuclear enriched abundant transcript 1 (NEAT1), a long noncoding RNA (lncRNA), is an oncogenic driver in HCC. In the present study, we investigated the significance of the NEAT1 gene in association with CD44 expression in liver CSCs of human HCC cell lines. The CSC properties were evaluated by spheroid culture, CSC marker expression, and sensitivity to anti-cancer drugs. The expression of both NEAT1 variant 1 (NEAT1v1) and variant 2 (NEAT1v2) as well as CD44 was significantly increased in the spheroid culture, compared with that in monolayer culture. Overexpression of Neat1v1, but not Neat1v2, enhanced the CSC properties, while knockout of the NEAT1 gene suppressed them. CD44 expression was increased by the overexpression of Neat1v1 and abrogated by NEAT1 knockout. The overexpression of NEAT1v1 restored the CSC properties and CD44 expression in NEAT1-knockout cells. NEAT1v1 expression in HCC tissues was correlated with poor prognosis and CD44 expression. These results suggest that NEAT1v1 is required for CD44 expression. To our surprise, NEAT1v1 also restored the CSC properties even in CD44-deficient cells, suggesting that NEAT1v1 maintains the properties of CSCs in a CD44-independent manner.

Evidence for Urate Uptake Through Monocarboxylate Transporter 9 Expressed in Mammalian Cells and Its Enhancement by Heat Shock.

Background: Monocarboxylate transporter 9 (MCT9), an orphan transporter member of the solute carrier family 16 (SLC16), possibly reabsorbs uric acid in the renal tubule and has been suggested by genome-wide association studies to be involved in the development of hyperuricemia and gout. In this study we investigated the mechanisms regulating the expression of human (h) MCT9, its degradation, and physiological functions. Methods and Results: hMCT9-FLAG was stably expressed in HEK293 cells and its degradation, intracellular localization, and urate uptake activities were assessed by pulse-chase analysis, immunofluorescence, and [14C]-urate uptake experiments, respectively. hMCT9-FLAG was localized on the plasma membrane as well as in the endoplasmic reticulum and Golgi apparatus. The proteasome inhibitors MG132 and lactacystine increased levels of hMCT9-FLAG protein expression with enhanced ubiquitination, prolonged their half-life, and decreased [14C]-urate uptake. [14C]-urate uptake was increased by both heat shock (HS) and the HS protein inducer geranylgeranylacetone (GGA). Both HS and GGA restored the [14C]-urate uptake impaired by MG132. Conclusions: hMCT9 does transport urate and is degraded by a proteasome, inhibition of which reduces hMCT9 expression on the cell membrane and urate uptake. HS enhanced urate uptake through hMCT9.

Determination of Minor and Trace Metals in Aluminum and Aluminum Alloys by ICP-AES; Evaluation of the Uncertainty and Limit of Quantitation from Interlaboratory Testing.

Minor and trace metals in aluminum and aluminum alloys have been determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) as an interlaboratory testing toward standardization. The trueness of the measured data was successfully investigated to improve the analytical protocols, using certified reference materials of aluminum. Their precision could also be evaluated, feasible to estimate the uncertainties separately. The accuracy (trueness and precision) of the data were finally in good agreement with the certified values and assigned uncertainties. Repeated measurements of aluminum solutions with different concentrations of the analytes revealed the relative standard deviations of the measurements with concentrations, thus enabling their limits of quantitation. They differed separately and also showed slightly higher values with an aluminum matrix than those without one. In addition, the upper limit of the detectable concentration of silicon with simple acid digestion was estimated to be 0.03 % in the mass fraction.

[A case of recurrence with liver metastasis after total gastrectomy for StageIA gastric carcinoma successfully treated with S-1/CDDP combination followed by S-1 chemotherapy].

A 72-year-old man underwent total gastrectomy for gastric carcinoma. Postoperative staging was Stage IA. One year after operation, abdominal CT revealed a metastatic tumor in the left lateral posterior segment of the liver. He was given S-1/ CDDP combination chemotherapy(S-1 120mg/body, day 1-21, CDDP 95mg/body, day 8)every 5weeks as first-line treatment. After 2 courses of the treatment, the liver tumor was not detected by PET-CT. The clinical response was assessed to be complete response. After total 5 courses of the treatment, we changed to a single administration of S-1(120mg/body, day 1- 14)every 3 or 4 weeks as second-line chemotherapy. The effect has been continued for 18 months after the initial metastasis. S-1/CDDP and S-1 chemotherapy are effective for metachronous liver metastasis from gastric carcinoma, although prognosis of the disease is poor.

Pre-administration of docetaxel protects against adriamycin-induced cardiotoxicity.

BACKGROUND: We have revealed in a pre-clinical study that the combination of adriamycin (ADR) and docetaxel (DOC) in which ADR was administered 12 h after DOC injection not only significantly reduced leukopenia and toxic death but also significantly increased the antitumor effect compared with the dosing schedule without an interval between each injection used commonly in clinical practice. The purpose of this study was to clarify in mice whether the toxic death caused by ADR was reduced by administering ADR after DOC injection when the doses and dosing-interval of ADR and DOC were changed. METHODS: ADR alone or a combination of ADR and DOC (ADR/DOC group in which both drugs were administered simultaneously or DOC-ADR group in which ADR was administered after DOC injection) was administered every 7 days in mice. RESULTS: When dosing intervals (0-24 h) were changed, there were no differences in survival rate among the 6, 12, and 24-h interval groups, although these groups showed significantly higher survival rate compared with the ADR/DOC group. When the dose of ADR (2.5-15 mg/kg) was changed, the survival rate was higher in all the DOC-ADR groups than the ADR alone groups. When the dose of DOC (3.125-12.5 mg/kg) was changed, DOC caused a dose-dependent reduction in toxic death. Although there was no striking difference in adverse effects between the ADR alone and DOC-ADR groups, the DOC-ADR group showed markedly attenuated increases in CPK-MB activity compared with the ADR alone group. CONCLUSIONS: We conclude that pre-administration of DOC may protect against ADR-induced toxic death and cardiotoxicity.

Transcriptional readthrough of Hox genes Ubx and Antp and their divergent post-transcriptional control during crustacean evolution.

Hox genes are in principle tandemly arranged in an order colinear with their order of expression along the anterior-posterior axis. Combinations of Hox proteins encode information that specifies the unique characteristics of axial regions in the metazoan body plan. The independent regulation of Hox genes achieved by differential promoter activity is essential for the expression of Hox proteins in distinct territories and thereby creating a full repertoire of Hox codes. Here we report the abundant expression of transcriptional readthrough products of two adjacent Hox genes, Ubx, and Antp, in five crustacean species of Branchiopoda and Malacostraca. Bicistronic mRNA places Antp under the control of the Ubx promoter, which is active in the posterior segments of two branchiopodans Daphnia and Artemia, and would normally reduce the complexity of Hox codes if translated. This does not occur, however, as the translational capability of the bicistronic mRNA is limited. In Daphnia, bicistronic Ubx/Antp mRNA produced no significant level of either UBX or ANTP. In Artemia, on the other hand, the bicistronic mRNA produced only UBX, and replaced the role of monocistronic Ubx mRNA. In this way, multiple post-transcriptional control mechanisms in two extant branchiopodans can be seen as preventing the potentially deleterious consequences of Hox gene fusion.

Therapeutic index by combination of adriamycin and docetaxel depends on dosing time in mice.

Although the combination of adriamycin and docetaxel showed a better cure rate against metastatic breast cancer, severe myelosuppression and cardiotoxicity were dose-limiting factors. The purpose of this study was to establish a suitable dosing schedule, based on a chronopharmacologic approach, to relieve severe adverse effects. In experiment 1, adriamycin or docetaxel was injected i.p. at 2, 6, 10, 14, 18, or 22 hours after light onset (HALO) to estimate toxicities. In experiment 2, the dosing time dependency of toxicity and pharmacokinetics were assessed in the combination of adriamycin and docetaxel. In addition, G2-M phase in myelocyte cells was determined in nontreated mice. Adverse effects caused by adriamycin were shown to be the worst at 2 HALO and the best at 14 HALO. On the other hand, docetaxel-induced adverse effects were more severe at 14 HALO than at 2 HALO. In the combination study, the D(2)-A(1)4 group, in which docetaxel was administered at 2 HALO followed by adriamycin at 14 HALO, showed the most toxicity relief of all the treated groups. In the pharmacokinetic study, the dosing time dependency of toxicities was not related to the daily variation of pharmacokinetics of adriamycin and docetaxel. A significant 24-hour rhythm of G2-M phase distribution was found in myelocyte cells of nontreated mice. The daily variation of leukopenia caused by docetaxel corresponded to the 24-hour rhythm of G2-M phase distribution. These findings reveal that the therapeutic index of the combined chemotherapy can be improved by administering adriamycin and docetaxel at the time when the most adverse effects are relieved in each drug.

Chronopharmacological study of antidepressants in forced swimming test of mice.

The influence of dosing time on the anti-immobility effect of antidepressants and mechanisms underlying this phenomenon were investigated in mice. In the forced swimming test (FST), the immobility time of mice treated with amitriptyline (15 mg/kg) and fluvoxamine (30 mg/kg) showed a significant 24-h rhythm. The anti-immobility effect of fluvoxamine in FST was potent at the early part of the dark phase without increasing locomotor activity. Concerning pharmacokinetics, although K(e) of fluvoxamine was approximately 1.3-fold higher in mice injected with fluvoxamine at 9:00 PM than at 9:00 AM, no dosing time dependence was demonstrated for either plasma or brain fluvoxamine concentration at 0.5 h after the drug injection. On the other hand, serotonin transporter (SERT) mRNA expression and 5-hydroxytryptamine (5-HT) uptake activity in the mouse midbrain showed significant time-dependent changes with higher levels during the dark phase and lower levels during the light phase. These results suggest that the reuptake of 5-HT might be more increased during the dark phase. Since the reuptake of 5-HT is inhibited almost completely by injection with 30 mg/kg fluvoxamine at any time, the extracellular 5-HT level may be more increased by the injection of fluvoxamine at the early part of the dark phase. The present results suggest that the anti-immobility effect of fluvoxamine in FST increases depending on dosing time. Furthermore, the time-dependent change of SERT mRNA expression and uptake activity in the midbrain is suggested to be the mechanism underlying the 24-h rhythm of anti-immobility effect of fluvoxamine.

Cloning of a quail homologue of hatching enzyme: its conserved function and additional function in egg envelope digestion.

The aim of the present study was to reveal molecular entities participating in the digestion of the egg envelope in the Japanese quail, Coturnix japonica. We isolated a 1,510-bp cDNA from extraembryonic tissues of developing embryos and designated it quail hatching enzyme (QHE) cDNA. The QHE cDNA was found to code a protein molecule comprising an astacin protease domain in the N-terminal half and a complement subcomponents C1r/C1s, Uegf, Bmp1 (CUB) domain in the C-terminal half. A phylogenetic analysis showed that QHE belonged to the hatching enzyme group and was distinct from other proteases in the astacin family. Northern blotting and in situ hybridization demonstrated that expression of the QHE mRNA occurred twice during the development: first in ectodermal cells of the yolk sac on days 0-5, then in those of the albumen sac on days 8-13. Zymography revealed that proteolytic activity in extracts of days 3-4 and 9-12 embryos appeared at the position of 40 kDa. Immunoblotting tests showed that anti-QHE antiserum stained a 40-kDa molecule in extracts of day 3 area vitellina. Anti-QHE antibody stained the ectodermal cells of the area opaca on days 0-1, those of the area vitellina of the yolk sac on days 2-5, and those of the albumen sac on days 9-12. The temporal and spatial expression pattern of QHE mRNA was closely associated with digestion of the vitelline membrane occurring on days 1-4, and with that of the egg white on days 9-12.

Characterization of oligosaccharide ligands expressed on SW1116 cells recognized by mannan-binding protein. A highly fucosylated polylactosamine type N-glycan.

Mannan-binding protein (MBP) is a C-type serum lectin and activates complement through the lectin pathway when it binds to ligand sugars such as mannose, N-acetylglucosamine, and fucose on microbes. In addition, the vaccinia virus carrying the human MBP gene was shown to exhibit potent growth inhibitory activity toward human colorectal carcinoma, SW1116, cells in nude mice. We have proposed calling this activity MBP-dependent cell-mediated cytotoxicity (MDCC) (Ma, Y., Uemura, K., Oka, S., Kozutsumi, Y., Kawasaki, N., and Kawasaki, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 371-375). In this study, the MBP ligands on the surface of SW1116 cells were characterized. Initial experiments involving plant lectins and anti-Lewis antibodies as inhibitors of MBP binding to SW1116 cells indicated that fucose plays a crucial role in the interaction. Subsequently, Pronase glycopeptides were prepared from whole cell lysates, and oligosaccharides were liberated by hydrazinolysis. After being tagged by pyridylamination, MBP ligand oligosaccharides were isolated with an MBP affinity column, and then their sequences were determined by mass spectrometry and tandem mass spectrometry after permethylation, in combination with endo-beta-galactosidase digestion and chemical defucosylation. The MBP ligands were shown to be large, multiantennary N-glycans carrying a highly fucosylated polylactosamine type structure. At the nonreducing termini, Le(b)/Le(a) or tandem repeats of the Le(a) structure prevail, a substantial proportion of which are attached via internal Le(x) or N-acetyllactosamine units to the trimannosyl core. The structures characterized are unique and distinct from those of other previously reported tumor-specific carbohydrate antigens. It is concluded that MBP requires clusters of tandem repeats of the Le(b)/Le(a) epitope for recognition.

Drosophila Nedd4 regulates endocytosis of notch and suppresses its ligand-independent activation.

BACKGROUND: Ligand-induced proteolytic cleavage and internalization of the plasma membrane receptor Notch leads to its activation. Ligand-independent, steady-state internalization of Notch, however, does not lead to activation. The mechanism by which downstream effectors discriminate between these bipartite modes of Notch internalization is not understood. Nedd4 is a HECT domain-containing E3 ubiquitin ligase that targets transmembrane receptors containing the PPSY motif for endocytosis. Deltex is a positive Notch signaling regulator that encodes a putative ubiquitin ligase of the ring finger type. RESULTS: We used the Drosophila system to show that Notch is ubiquitinated and destabilized by Nedd4 in a manner requiring the PPSY motif in the Notch intracellular domain. Loss of Nedd4 function dominantly suppresses the Notch and Deltex mutant phenotypes, and its hyperactivation attenuates Notch activity. In tissue culture cells, the dominant-negative form of Nedd4 blocks steady-state Notch internalization and activates Notch signaling independently of ligand binding. This effect was further potentiated by Deltex. Nedd4 destines Deltex for degradation in a Notch-dependent manner. CONCLUSIONS: Nedd4 antagonizes Notch signaling by promoting degradation of Notch and Deltex. This Nedd4 function may be important for protecting unstimulated cells from sporadic activation of Notch signaling.

Influence of dosing schedule on toxicity and antitumor effects of a combination of adriamycin and docetaxel in mice.

PURPOSE: Although the combination of Adriamycin (ADR) and docetaxel (DOC) showed a better cure rate against metastatic breast cancer in a clinical study, severe myelosuppression and cardiotoxicity were dose-limiting factors. The purpose of this study was to establish the most suitable dosing schedule to relieve severe adverse effects and improve the antitumor effects. EXPERIMENTAL DESIGN: Both ADR and DOC were administered simultaneously in the simultaneous-dosing group (ADR/DOC), whereas in the intermittent-dosing groups (ADR-DOC and DOC-ADR), the second drug was administered 12 h after the first drug. Leukocyte counts and survival were measured to estimate adverse effects. After administration, ADR and DOC concentrations in blood, myelocyte cells, and heart were determined. To clarify the antitumor effect, tumor growth was measured in Ehrlich-cell-bearing mice after the initiation of drug injections. RESULTS: The simultaneous-dosing group showed severe leukopenia compared with the saline-treated group. However, the toxicity was reduced in the intermittent-dosing groups. The DOC-ADR group showed the best survival rate in the dosing groups. In the pharmacokinetic study, ADR and DOC concentrations in plasma, myelocyte cells, and the heart were markedly higher in the simultaneous-dosing group than the intermittent-dosing groups. These results indicate that pharmacokinetic interactions may contribute to the change in leukopenia induced by concurrent administration of ADR and DOC. The antitumor effect in the DOC-ADR group was the highest in the dosing groups. CONCLUSIONS: In the present study, the findings suggest that ADR administered 12 h after DOC injection (DOC-ADR group) not only inhibits tumor growth more strongly but also significantly reduces leukopenia compared with results for the simultaneous-dosing (ADR/DOC) group and significantly reduced the number of toxic deaths compared with the other groups.