Gestational Outcomes and Birth Weight in Japanese Women at the Upper and Lower limits of the Normal BMI range.

To examine the outcome of gestational blood pressure and birth weight in women with normal pre-pregnancy BMI (18.5-25 kg/m2) who are at the lower and upper limits of this range, i.e., slightly underweight or slightly overweight. Overall, 2,038 Japanese women with low -risk who had delivered during January 2014-December 2016 were classified according to their pre-pregnancy BMI: underweight (< 18.5 kg/m2), slightly underweight (18.5≤BMI<21 kg/m2), normal (21≤BMI<23 kg/m2), slightly overweight (23≤BMI<25 kg/m2) and overweight (≤ 25 kg/m2). Their blood pressure during each trimester and birth weight was evaluated. The slightly overweight group showed a significantly higher blood pressure than the underweight and slightly underweight groups. Birth weight was lower in the slightly underweight than in the slightly overweight group (p<0.01). The incidence rate of "heavy for dates" (HFD) infants was significantly higher in the slightly overweight and overweight groups than in the other groups (p<0.05 and p<0.01, respectively). Weight gain of < 7 kg significantly increased the rate of "light for dates" (LFD) infants, while a weight gain of ≥13 kg significantly increased the rate of HFD infants (p<0.05 and p<0.01, respectively). Blood pressure during pregnancy was ssociated with pre-pregnancy BMI. The birth weight of infants of low-risk pregnant women is affected by both pre-pregnancy BMI and gestational weight gain.

Candidemia in COVID-19 treated with corticosteroids and tocilizumab.

COVID-19 patients treated with anti-inflammatory drugs are rarely complicated by candidemia. Since immunosuppressive therapy can blunt inflammatory reactions, clinicians should actively survey latent candidemia during severe COVID-19 treatment.

Spred2-deficiency enhances the proliferation of lung epithelial cells and alleviates pulmonary fibrosis induced by bleomycin.

The mitogen-activated protein kinase (MAPK) pathways are involved in many cellular processes, including the development of fibrosis. Here, we examined the role of Sprouty-related EVH-1-domain-containing protein (Spred) 2, a negative regulator of the MAPK-ERK pathway, in the development of bleomycin (BLM)-induced pulmonary fibrosis (PF). Compared to WT mice, Spred2-/- mice developed milder PF with increased proliferation of bronchial epithelial cells. Spred2-/- lung epithelial cells or MLE-12 cells treated with spred2 siRNA proliferated faster than control cells in vitro. Spred2-/- and WT macrophages produced similar levels of TNFα and MCP-1 in response to BLM or lipopolysaccharide and myeloid cell-specific deletion of Spred2 in mice had no effect. Spred2-/- fibroblasts proliferated faster and produced similar levels of MCP-1 compared to WT fibroblasts. Spred2 mRNA was almost exclusively detected in bronchial epithelial cells of naïve WT mice and it accumulated in approximately 50% of cells with a characteristic of Clara cells, 14 days after BLM treatment. These results suggest that Spred2 is involved in the regulation of tissue repair after BLM-induced lung injury and increased proliferation of lung bronchial cells in Spred2-/- mice may contribute to faster tissue repair. Thus, Spred2 may present a new therapeutic target for the treatment of PF.

MicroRNA biomarkers for chemical hazard screening identified by RNA deep sequencing analysis in mouse embryonic stem cells.

We investigated the responses of microRNAs (miRNAs) using mouse embryonic stem cells (mESCs) exposed to nine chemicals (bis(2-ethylhexyl)phthalate, p-cresol, p-dichlorobenzene, phenol, pyrocatecol, chloroform, tri-n-butyl phosphate, trichloroethylene, and benzene), which are listed as "Class I Designated Chemical Substances" from the Japan Pollutant Release and Transfer Register. Using deep sequencing analysis (RNA-seq), several miRNAs were identified that show a substantial response to general chemical toxicity (i.e., to these nine chemicals considered as a group) and several miRNA biomarkers that show a substantial and specific response to benzene. The functions of the identified miRNAs were investigated in accordance with Gene Ontology terms of their predicted target genes, indicating regulation of cellular processes. We compared the results with those for the long non-coding RNAs (ncRNAs) and mRNAs reported in our previous studies in addition to previously identified miRNAs that are either up- or down-regulated in response to the benzene as stimuli. We also observed that the changes in expression of miRNAs were smaller than those for long ncRNAs and mRNAs. Taken together the current and previous results revealed that toxic chemical stimuli regulate the expression of miRNAs. We believe that the use of miRNAs, including the thus identified miRNAs, as biomarkers contribute to predicting the potential toxicity of particular chemicals or identifying human individuals that have been exposed to chemical hazards.

Identification of RNA biomarkers for chemical safety screening in neural cells derived from mouse embryonic stem cells using RNA deep sequencing analysis.

Chemical safety screening requires the development of more efficient assays that do not involve testing in animals. In vitro cell-based assays are among the most appropriate alternatives to animal testing for screening of chemical toxicity. Most studies performed to date made use of mRNAs as biomarkers. Recent studies have however indicated the presence of many unannotated non-coding RNAs (ncRNAs) in the transcriptome that do appear to encode proteins. In the present study, we performed whole-transcriptome sequencing analysis (RNA-Seq) to identify novel RNA biomarkers, including ncRNAs, which showed marked responses to the toxicity of nine chemicals. Chemical safety screening was performed in cell-based assays using mouse embryonic stem cell (mESC)-derived neural cells. Marked responses in the expression of some ncRNAs to the chemical compounds were observed. The results of the present study suggested that ncRNAs may be useful in chemical safety screening as novel RNA biomarkers.

Cancer Cell-Derived Granulocyte-Macrophage Colony-Stimulating Factor Is Dispensable for the Progression of 4T1 Murine Breast Cancer.

We previously reported that 4T1 murine breast cancer cells produce GM-CSF that up-regulates macrophage expression of several cancer promoting genes, including Mcp-1/Ccl2, Ccl17 and Rankl, suggesting a critical role of cancer cell-derived GM-CSF in cancer progression. Here, we attempted to define whether 4T1 cell-derived GM-CSF contributes to the expression of these genes by 4T1tumors, and their subsequent progression. Intraperitoneal injection of anti-GM-CSF neutralizing antibody did not decrease the expression of Mcp-1, Ccl17 or Rankl mRNA by 4T1 tumors. To further examine the role of cancer cell-derived GM-CSF, we generated GM-CSF-deficient 4T1 cells by using the Crisper-Cas9 system. As previously demonstrated, 4T1 cells are a mixture of cells and cloning of cells by itself significantly reduced tumor growth and lung metastasis. By contrast, GM-CSF-deficiency did not affect tumor growth, lung metastasis or the expression of these chemokine and cytokine genes in tumor tissues. By in-situ hybridization, the expression of Mcp-1 mRNA was detected in both F4/80-expressing and non-expressing cells in tumors of GM-CSF-deficient cells. These results indicate that cancer cell-derived GM-CSF is dispensable for the tuning of the 4T1 tumor microenvironment and the production of MCP-1, CCL17 or RANKL in the 4T1 tumor microenvironment is likely regulated by redundant mechanisms.

Effect of methyl p-hydroxybenzoate on the culture of mammalian cell.

Several chemicals, such as methyl p-hydroxybenzoate (MHB), have been widely used as preservatives in the water baths of CO2 incubators used for mammalian cell culture, and they are not considered to produce any biological effects. However, no detailed analyses of the effects of these compounds on cultured cells have been reported. In this study, we thus examined whether MHB in the incubator water bath affects cell viability or genome-wide gene expression in mouse embryonic stem cells under control conditions [using only dimethyl sulfoxide (DMSO) in the culture medium] and under chemical-treated conditions using benzene and chloroform; conditions that simulate a cell-based toxicity assay. We found that (i) MHB significantly altered cell growth rate, and (ii) MHB affected gene expression levels related to pathways that modulate cell growth and basic molecular processes, not only under control conditions but also the chemical-treated conditions. Furthermore, Gene Ontology term analyses revealed that the effects of MHB cannot be accounted for by subtracting the gene expression pattern in the control conditions from that in the chemical-treated conditions. Thus, we suggest that the use of MHB or other preservatives in a CO2 incubator water bath is reconsidered in terms of potential confounding effects on cultured cells.

Identification of RNA biomarkers for chemical safety screening in mouse embryonic stem cells using RNA deep sequencing analysis.

Although it is not yet possible to replace in vivo animal testing completely, the need for a more efficient method for toxicity testing, such as an in vitro cell-based assay, has been widely acknowledged. Previous studies have focused on mRNAs as biomarkers; however, recent studies have revealed that non-coding RNAs (ncRNAs) are also efficient novel biomarkers for toxicity testing. Here, we used deep sequencing analysis (RNA-seq) to identify novel RNA biomarkers, including ncRNAs, that exhibited a substantial response to general chemical toxicity from nine chemicals, and to benzene toxicity specifically. The nine chemicals are listed in the Japan Pollutant Release and Transfer Register as class I designated chemical substances. We used undifferentiated mouse embryonic stem cells (mESCs) as a simplified cell-based toxicity assay. RNA-seq revealed that many mRNAs and ncRNAs responded substantially to the chemical compounds in mESCs. This finding indicates that ncRNAs can be used as novel RNA biomarkers for chemical safety screening.

Short-lived long non-coding RNAs as surrogate indicators for chemical exposure and LINC00152 and MALAT1 modulate their neighboring genes.

Whole transcriptome analyses have revealed a large number of novel long non-coding RNAs (lncRNAs). Although accumulating evidence demonstrates that lncRNAs play important roles in regulating gene expression, the detailed mechanisms of action of most lncRNAs remain unclear. We previously reported that a novel class of lncRNAs with a short half-life (t1/2 < 4 h) in HeLa cells, termed short-lived non-coding transcripts (SLiTs), are closely associated with physiological and pathological functions. In this study, we focused on 26 SLiTs and nuclear-enriched abundant lncRNA, MALAT1(t1/2 of 7.6 h in HeLa cells) in neural stem cells (NSCs) derived from human induced pluripotent stem cells, and identified four SLiTs (TUG1, GAS5, FAM222-AS1, and SNHG15) that were affected by the following typical chemical stresses (oxidative stress, heavy metal stress and protein synthesis stress). We also found the expression levels of LINC00152 (t1/2 of 2.1 h in NSCs), MALAT1 (t1/2 of 1.8 h in NSCs), and their neighboring genes were elevated proportionally to the chemical doses. Moreover, we confirmed that the overexpression of LINC00152 or MALAT1 upregulated the expressions of their neighboring genes even in the absence of chemical stress. These results reveal that LINC00152 and MALAT1 modulate their neighboring genes, and thus provide a deeper understanding of the functions of lncRNAs.

Characterization of Bombyx mori mitochondrial transcription factor A, a conserved regulator of mitochondrial DNA.

In the present study, we initially cloned and characterized a mitochondrial transcription factor A (Tfam) homologue in the silkworm, Bombyx mori. Bombyx mori TFAM (BmTFAM) localized to mitochondria in cultured silkworm and human cells, and co-localized with mtDNA nucleoids in human HeLa cells. In an immunoprecipitation analysis, BmTFAM was found to associate with human mtDNA in mitochondria, indicating its feature as a non-specific DNA-binding protein. In spite of the low identity between BmTFAM and human TFAM (26.5%), the expression of BmTFAM rescued mtDNA copy number reductions and enlarged mtDNA nucleoids in HeLa cells, which were induced by human Tfam knockdown. Thus, BmTFAM compensates for the function of human TFAM in HeLa cells, demonstrating that the mitochondrial function of TFAM is highly conserved between silkworms and humans. BmTfam mRNA was strongly expressed in early embryos. Through double-stranded RNA (dsRNA)-based RNA interference (RNAi) in silkworm embryos, we found that the knockdown of BmTFAM reduced the amount of mtDNA and induced growth retardation at the larval stage. Collectively, these results demonstrate that BmTFAM is a highly conserved mtDNA regulator and may be a good candidate for investigating and modulating mtDNA metabolism in this model organism.

Enhancement of Skin Penetration of Hydrophilic and Lipophilic Compounds by pH-sensitive Liposomes.

PURPOSE: Enhance skin penetration of hydrophilic and lipophilic compounds using liposomes that are responsible to the pH of the skin surface. METHODS: pH-sensitive liposomes were prepared by a thin layer and freeze-thaw method with dioleoyl phosphatidyl ethanolamine and cholesteryl hemisuccinate. Liposomal fusion with stratum corneum lipid liposomes was measured using fluorescence resonance energy transfer. Particle diameter and zeta potential of the liposomes after fusion were measured by dynamic light scattering and electrophoresis. RESULTS: Under neutral pH conditions, the diameter of the pH-sensitive liposomes was 130 nm and their zeta potential was -70 mV. In weakly acidic conditions, the diameter was larger than 3,000 nm and the zeta potential was -50 mV. In contrast, the particle diameter and the zeta potential of the non-pH-sensitive liposomes remained constant under various pH conditions. A skin penetration study was performed on hairless mice skin using vertical diffusion cells, showing that the fusion ability of pH-sensitive liposomes was higher than that of non-pH-sensitive liposomes. In the skin penetration study was carried out using hydrophilic (calcein) and lipophilic (N-(7-nitrobenz- 2-oxa-1,3-diazol-4yl)-PE) (NBD-PE) model compounds which were applied to the skin with pH-sensitive liposomes as carrier. The fluorescent compounds contained within the pH-sensitive liposomes permeated the skin more effectively than those within non-pH-sensitive liposomes, and this ability was further enhanced with the lipophilic compound. CONCLUSION: These studies suggest that pH-sensitive liposomes have potential as an important tool for delivery of compounds into the skin.

[Solitary pigment epithelial lesion accompanied by uveal effusion with bullous retinal detachment].

PURPOSE: To report a case of solitary pigment epithelial lesion accompanied by uveal effusion (UE) with bullous retinal detachment (RD). CASE: A 63-year-old man was referred to our hospital for RD in his right eye. Best corrected visual acuity was 20/20 and intraocular pressure was 14 mmHg in the right eye. Fundus examination showed UE in the entire peripheral zone with bullous RD in the inferior retina and a grayish-white placoid lesion with indistinct border at the level of the retinal pigment epithelium at the temporal area near the macula in the right eye. No retinal tear was found, and anterior chamber depth and axial length were within the normal range. Fluorescein angiography indicated dye leakage from the placoid lesion, but pooling of dye was not intensive. Since posterior scleritis was not excluded, a systemic corticosteroid was administered but the UE with bullous RD did not improve, thererfore, photocoagulation for the placoid lesion was performed. This gradually ameliorated the UE with bullous RD, and it resolved at 4 months after the first visit without any further recurrence. CONCLUSION: Solitary pigment epithelial lesion can cause UE with bullous RD as in multifocal posterior pigment epitheliopathy (MPPE).

Photobiocatalyzed asymmetric reduction of ketones using Chlorella sp. MK201.

Aromatic ketones were reduced using suspension culture of Chlorella sp. MK201 under fluorescent light illumination producing the corresponding chiral alcohols in high yields with excellent enantiomeric excess (ee). For example, 2',3',4',5',6'-pentafluoroacetophenone at 0.25 mg/ml was converted to the corresponding (S)-alcohol in 80 % yield with >99 % ee by 1 mg dry wt of Chlorella/ml in 12 h illumination (2,000 lux).

The rapid efficacy of abatacept in a patient with rheumatoid vasculitis.

We report a case of rheumatoid vasculitis (RV) that responded well to abatacept, a cytotoxic T lymphocyte-associated antigen 4 (CTLA4)-immunoglobulin fusion protein. A 38-year-old woman developed RV despite treatment with methotrexate and tumor necrosis factor (TNF) inhibitors. The effects of steroid therapy, immunoabsorption plasmapheresis, and interleukin-6 inhibitor were insufficient, however, administration of abatacept rapidly improved her clinical symptoms with almost normalization of the immunological findings. This is the first published case report of the successful treatment of RV with abatacept.

15-Deoxy-Δ¹²,¹4 prostaglandin J2 reduces the formation of atherosclerotic lesions in apolipoprotein E knockout mice.

AIM: 15-deoxy-Δ¹²,¹4 prostaglandin J2 (15d-PGJ2) is a ligand of peroxisome proliferator-activated receptor γ (PPARγ) having diverse effects such as the differentiation of adipocytes and atherosclerotic lesion formation. 15d-PGJ2 can also regulate the expression of inflammatory mediators on immune cells independent of PPARγ. We investigated the antiatherogenic effect of 15d-PGJ2. METHODS: We fed apolipoprotein (apo) E-deficient female mice a Western-type diet from 8 to 16 wk of age and administered 1 mg/kg/day 15d-PGJ2 intraperitoneally. We measured atherosclerotic lesions at the aortic root, and examined the expression of macrophage and inflammatory atherosclerotic molecules by immunohistochemical and real-time PCR in the lesion. RESULTS: Atherosclerotic lesion formation was reduced in apo E-null mice treated with 15d-PGJ2, as compared to in the controls. Immunohistochemical and real-time PCR analyses showed that the expression of MCP-1, TNF-α, and MMP-9 in atherosclerotic lesions was significantly decreased in 15d-PGJ2 treated mice. The 15d-PGJ2 also reduced the expression of macrophages and RelA mRNA in atherosclerotic lesions. CONCLUSION: This is the first report 15d-PGJ2, a natural PPARγ agonist, can improve atherosclerotic lesions in vivo. 15d-PGJ2 may be a beneficial therapeutic agent for atherosclerosis.

Reduction of exogenous ketones depends upon NADPH generated photosynthetically in cells of the cyanobacterium Synechococcus PCC 7942.

Effective utilization of photosynthetic microorganisms as potential biocatalysts is favorable for the production of useful biomaterials and the reduction of atmospheric CO2. For example, biocatalytic transformations are used in the synthesis of optically active alcohols. We previously found that ketone reduction in cells of the cyanobacterium Synechococcus PCC 7942 is highly enantioselective and remarkably enhanced under light illumination. In this study, the mechanism of light-enhanced ketone reduction was investigated in detail using several inhibitors of photosynthetic electron transport and of enzymes of the Calvin cycle. It is demonstrated that light intensity and photosynthesis inhibitors significantly affect the ketone reduction activity in Synechococcus. This indicates that the reduction correlates well with photosynthetic activity. Moreover, ketone reduction in Synechococcus specifically depends upon NADPH and not NADH. These results also suggest that cyanobacteria have the potential to be utilized as biocatalytic systems for direct usage of light energy in various applications such as syntheses of useful compounds and remediation of environmental pollutants.

[Juvenile delinquents' loneliness as represented on the Doll Location Test].

This study examined the validity of the indices of Hatta's Doll Location Test (DLT) to represent the interpersonal structure of juvenile delinquents. Japanese juvenile delinquents (N = 215) in detention were asked to fill out the Differential Loneliness Scale and to represent their typical interpersonal relations using the DLT. The results showed that the presence, the order, the distance, and the height of the figures were related to loneliness. However, the direction of placing the figures did not show the expected relationship with loneliness. Furthermore, the relationships between the indices and loneliness were different according to the type of person represented. The results suggest that distance was valid as an index of cohesion, although height could discriminate the intimate group from the others.

[Testicular tumor presenting as acute scrotum with infection caused by gas-producing bacteria].

A 46-year-old male patient visited our hospital with the chief complaint of a right scrotal swelling and pain. The enlarged scrotum was 8 cm in diameter with redness of the skin of the right scrotum. There was a firm mass in the scrotum with marked tenderness. The patient's body temperature was 38.0C, and blood tests showed increased inflammatory markers. The results of computed tomography and magnetic resonance imaging showed that the enlarged scrotum was filled with gas and there was a mass partially composed of fat inside it. The patient was treated with antibiotics, but the fever persisted and the inflammatory markers further increased. The size of the right scrotum gradually increased. A right high inguinal orchiectomy was performed to control the inflammation and makea diagnosis. Thehistopathological diagnosis was necrosis of seminoma, and culture of thene crotic tissuewas positivefor Clostridium. Thepre sent caseappe ars to bethefirst caseof a testicular tumor associated with acute scrotum due to tumor growth, necrosis and infection caused by gas-producing bacteria.

[A case of chronic expanding hematoma in retroperitoneal space].

A 34-year-old female patient visited a nearby hospital with a chief complaint of right flank pain and decreased weight. Computed tomography showed a right retroperitoneal mass 10 cm in diameter on the right kidney, displacing the liver and the right kidney. The patient was referred to Kawasaki Municipal Hospital for further evaluation. The mass was suspected to be chronic expanding hematoma or neurogenic tumor of renal capsule origin. A retroperitoneal tumorectomy was performed with a right subcostal incision. A mass was noted in the smooth capsule. The mass was easily removed from the right renal capsule. However, there was significant adhesion between the mass and the peritoneum as well as the liver capsule. Therefore, a partial hepatectomy was needed for complete resection of the mass including the capsule. A fibrous capsule was noted and most of the mass was blood clot-like tissue. The histopathological diagnosis was chronic expanding hematoma with no malignancy. A retroperitoneal chronic expanding hematoma has very rarely been reported.

Analysis of two gene clusters involved in the degradation of 4-fluorophenol by Arthrobacter sp. strain IF1.

Arthrobacter sp. strain IF1 is able to grow on 4-fluorophenol (4-FP) as a sole source of carbon and energy. To clone the 4-FP degradation genes, DNA libraries were constructed and screened with a probe obtained by PCR using primers designed on the basis of conserved regions of aromatic two-component monooxygenases. Sequencing of positive clones yielded two gene clusters, each harboring a gene encoding a monooxygenase with high sequence similarity to the oxygenase component of 4-nitrophenol and 4-chlorophenol monooxygenase systems. Both these monooxygenase genes were differentially expressed during growth on 4-FP, as revealed by Northern blotting and reverse transcription-PCR. One cluster also contained a gene for a flavin reductase. The monooxygenase and reductase were purified from Escherichia coli cells expressing the corresponding genes, and together they catalyzed NADH-dependent hydroxylation and dehalogenation of 4-halophenols. The results indicate that strain IF1 transforms 4-FP to hydroquinone by a two-component monooxygenase system of which one component provides reduced flavin adenine dinucleotide at the expense of NADH and the other catalyzes para-hydroxylation of 4-FP and other 4-substituted phenols.