Helicobacter cinaedi is the most common enterohepatic Helicobacter species that causes bacteremia in humans, but its pathogenicity is unclear. Here, we investigated the possible association of H. cinaedi with atherosclerosis in vivo and in vitro. We found that H. cinaedi infection significantly enhanced atherosclerosis in hyperlipidaemic mice. Aortic root lesions in infected mice showed increased accumulation of neutrophils and F4/80(+) foam cells, which was due, at least partly, to bacteria-mediated increased expression of proinflammatory genes. Although infection was asymptomatic, detection of cytolethal distending toxin RNA of H. cinaedi indicated aorta infection. H. cinaedi infection altered expression of cholesterol receptors and transporters in cultured macrophages and caused foam cell formation. Also, infection induced differentiation of THP-1 monocytes. These data provide the first evidence of a pathogenic role of H. cinaedi in atherosclerosis in experimental models, thereby justifying additional investigations of the possible role of enterohepatic Helicobacter spp. in atherosclerosis and cardiovascular disease.

18F-FDG PET/CT has been used as an indicator of chemotherapy effects, but cancer cells can remain even when no FDG uptake is detected, indicating the importance of exploring other metabolomic pathways. Therefore, we explored the amino acid metabolism, including L-type amino acid transporter-1 (LAT1), in breast cancer tissues and clarified the role of LAT1 in therapeutic resistance and clinical outcomes of patients. We evaluated LAT1 expression before and after neoadjuvant chemotherapy and examined the correlation of glucose uptake using FDG-PET with the pathological response of patients. It revealed that LAT1 levels correlated with proliferation after chemotherapy, and amino acid and glucose metabolism were closely correlated. In addition, LAT1 was considered to be involved in treatment resistance and sensitivity only in luminal type breast cancer. Results of in vitro analyses revealed that LAT1 promoted amino acid uptake, which contributed to energy production by supplying amino acids to the TCA cycle. However, in MCF-7 cells treated with chemotherapeutic agents, oncometabolites and branched-chain amino acids also played a pivotal role in energy production and drug resistance, despite decreased glucose metabolism. In conclusion, LAT1 was involved in drug resistance and could be a novel therapeutic target against chemotherapy resistance in luminal type breast cancer.

The Drosophila behavior/human splicing protein family is involved in numerous steps of gene regulation. In humans, this family consists of three proteins: SFPQ, PSPC1, and NONO. Hemizygous loss-of-function (LoF) variants in NONO cause a developmental delay with several complications (e.g., distinctive facial features, cardiac symptoms, and skeletal symptoms) in an X-linked recessive manner. Most of the reported variants have been LoF variants, and two missense variants have been reported as likely deleterious but with no functional validation. We report three individuals from two families harboring an identical missense variant that is located in the nuclear localization signal, NONO: NM_001145408.2:c.1375C > G p.(Pro459Ala). All of them were male and the variant was inherited from their asymptomatic mothers. Individual 1 was diagnosed with developmental delay and cardiac phenotypes (ventricular tachycardia and dilated cardiomyopathy), which overlapped with the features of reported individuals having NONO LoF variants. Individuals 2 and 3 were monozygotic twins. Unlike in Individual 1, developmental delay with autistic features was the only symptom found in them. A fly experiment and cell localization experiment showed that the NONO variant impaired its proper intranuclear localization, leading to mild LoF. Our findings suggest that deleterious NONO missense variants should be taken into consideration when whole-exome sequencing is performed on male individuals with developmental delay with or without cardiac symptoms.

Influenza virus is rich in variation and mutations. It would be very convenient for virus detection and isolation to histochemically detect viral infection regardless of variation and mutations. Here, we established a histochemical imaging assay for influenza virus sialidase activity in living cells by using a new fluorescent sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac). The BTP3-Neu5Ac assay histochemically visualized influenza virus-infected cells regardless of viral hosts and subtypes. Influenza virus neuraminidase-expressed cells, viral focus formation, and virus-infected locations in mice lung tissues were easily, rapidly, and sensitively detected by the BTP3-Neu5Ac assay. Histochemical visualization with the BTP3-Neu5Ac assay is extremely useful for detection of influenza viruses without the need for fixation or a specific antibody. This novel assay should greatly improve the efficiency of detection, titration, and isolation of influenza viruses and might contribute to research on viral sialidase.

Breast cancer is a hormone-dependent cancer and usually treated with endocrine therapy using aromatase inhibitors or anti-estrogens such as tamoxifen. A majority of breast cancer, however, will often fail to respond to endocrine therapy. In the present study, we explored miRNAs associated with endocrine therapy resistance in breast cancer. High-throughput miRNA sequencing was performed using RNAs prepared from breast cancer MCF-7 cells and their derivative clones as endocrine therapy resistant cell models, including tamoxifen-resistant (TamR) and long-term estrogen-deprived (LTED) MCF-7 cells. Notably, miR-21 was the most abundantly expressed miRNA in MCF-7 cells and overexpressed in TamR and LTED cells. We found that miR-378a-3p expression was downregulated in TamR and LTED cells as well as in clinical breast cancer tissues. Additionally, lower expression levels of miR-378a-3p were associated with poor prognosis for tamoxifen-treated patients with breast cancer. GOLT1A was selected as one of the miR-378a-3p candidate target genes by in silico analysis. GOLT1A was overexpressed in breast cancer specimens and GOLT1A-specific siRNAs inhibited the growth of TamR cells. Low GOLT1A levels were correlated with better survival in patients with breast cancer. These results suggest that miR-378a-3p-dependent GOLT1A expression contributes to the mechanisms underlying breast cancer endocrine resistance.

Sialidase cleaves sialic acid residues from a sialoglycoconjugate: oligosaccharides, glycolipids and glycoproteins that contain sialic acid. Histochemical imaging of the mouse pancreas using a benzothiazolylphenol-based sialic acid derivative (BTP3-Neu5Ac), a highly sensitive histochemical imaging probe used to assess sialidase activity, showed that pancreatic islets have intense sialidase activity. The sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA) remarkably enhances glutamate release from hippocampal neurons. Since there are many similar processes between synaptic vesicle exocytosis and secretory granule exocytosis, we investigated the effect of DANA on insulin release from β-cells. Insulin release was induced in INS-1D cells by treatment with 8.3 mM glucose, and the release was enhanced by treatment with DANA. In a mouse intraperitoneal glucose tolerance test, the increase in serum insulin levels was enhanced by intravenous injection with DANA. However, under fasting conditions, insulin release was not enhanced by treatment with DANA. Calcium oscillations induced by 8.3 mM glucose treatment of INS-1D cells were not affected by DANA. Blood insulin levels in sialidase isozyme Neu3-deficient mice were significantly higher than those in WT mice under ad libitum feeding conditions, but the levels were not different under fasting conditions. These results indicate that DANA is a glucose-dependent potentiator of insulin secretion. The sialidase inhibitor may be useful for anti-diabetic treatment with a low risk of hypoglycemia.

The oral cavity is an entrance for respiratory viruses, such as influenza. Recently, saliva has been shown to exert both antimicrobial and antiviral activities. Thus, saliva may be a biological factor that contributes to the prevention of influenza infection. However, the actual salivary anti-influenza A virus (IAV) activity in individuals and its determinant factors are unknown. By assessing individual variations in salivary anti-IAV activity in 92 people using an established new high-throughput system in this study, we found that the anti-IAV activity varied widely between individuals and showed a significant positive correlation with protein-bound sialic acid (BSA) level (ρ = 0.473; p < 0.001). Furthermore, the anti-IAV activity of saliva with enzymatically reduced BSA content was significantly lower. These results indicate that BSA is a direct regulator of salivary anti-IAV activity and is a determinant of individual differences. Additionally, after comparing the anti-IAV activity across the groups by age, anti-IAV activity in young people (aged 5-19 years) were lower than in adults aged 20-59 years and elderly people aged 60-79 years. Our study suggests that BSA levels in saliva may be important in preventing influenza infection.

Microsomal triglyceride transfer protein (MTP) is a unique lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins by the liver and intestine. Previous studies in mice identified a splice variant of MTP with an alternate first exon. Splice variants of human MTP have not been reported. Using PCR approaches we have identified two splice variants in human tissues, which we have named MTP-B and MTP-C. MTP-B has a unique first exon (Ex1B) located 10.5 kb upstream of the first exon (Ex1A) for canonical MTP (MTP-A); MTP-C contains both first exons for MTP-A and MTP-B. MTP-B was found in a number of tissues, whereas MTP-C was prominent in brain and testis. MTP-B does not encode a protein; MTP-C encodes the same protein encoded by MTP-A, although MTP-C translation is strongly inhibited by regulatory elements within its 5'-UTR. Using luciferase assays, we demonstrate that the promoter region upstream of exon 1B is quite adequate to drive expression of MTP. We conclude that alternate splicing plays a key role in regulating cellular MTP levels by introducing distinct promoter regions and unique 5'-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP activity.

Amyloid β-protein (Aβ42) oligomerization is an early event in Alzheimer's disease (AD). Current diagnostic methods using sequence-specific antibodies against less toxic fibrillar and monomeric Aβ42 run the risk of overdiagnosis. Hence, conformation-specific antibodies against neurotoxic Aβ42 oligomers have garnered much attention for developing more accurate diagnostics. Antibody 24B3, highly specific for the toxic Aβ42 conformer that has a turn at Glu22 and Asp23, recognizes a putative Aβ42 dimer, which forms stable and neurotoxic oligomers more potently than the monomer. 24B3 significantly rescues Aβ42-induced neurotoxicity, whereas sequence-specific antibodies such as 4G8 and 82E1, which recognizes the N-terminus, do not. The ratio of toxic to total Aβ42 in the cerebrospinal fluid of AD patients is significantly higher than in control subjects as measured by sandwich ELISA using antibodies 24B3 and 82E1. Thus, 24B3 may be useful for AD diagnosis and therapy.

In vivo gene delivery is required for studying the cellular and molecular mechanisms of various biological events. Virus-mediated gene transfer or generation of transgenic animals is widely used; however, these methods are time-consuming and expensive. Here we show an improved electroporation technique for acute gene delivery into the adult mouse brain. Using a syringe-based microelectrode, local DNA injection and the application of electric current can be performed simultaneously; this allows rapid and efficient gene transduction of adult non-neuronal cells. Combining this technique with various expression vectors that carry specific promoters resulted in targeted gene expression in astrocytic cells. Our results constitute a powerful strategy for the genetic manipulation of adult brains in a spatio-temporally controlled manner.

Mature mammalian brains consist of variety of neuronal and non-neuronal cell types, which are progressively generated from embryonic neural progenitors through the embryonic and postnatal periods. However, it remains unknown whether all embryonic progenitors equivalently contribute to multiple cell types, or individual neural progenitors have variable potentials to generate specific cell types in a stochastic manner. Here, we performed population-level tracing of mouse embryonic neural progenitors by using Tol2-mediated genome integration vectors. We identified that neural progenitors in early embryonic stages predominantly contribute to cortical or subcortical neurons than astrocytes, ependymal cells, and neuroblasts in the postnatal brain. Notably, neurons and astrocytes were cumulatively labeled by the increase of total labeled cells, suggesting constant neurogenic and gliogenic potentials of individual neural progenitors. On the contrary, numbers of labeled ependymal cell are more fluctuated, implicating intrinsic variability of progenitor potentials for ependymal cell generation. Differential progenitor potentials that contribute to neurons, astrocytes, and ependymal cells were also detected in the developing avian pallium. Our data suggest evolutionary conservations of coherent and variable potentials of neural progenitors that generate multiple cell types in the developing amniote brain.

To date, various human disease models in small fish-such as medaka (Oryzias lapties)-have been developed for medical and pharmacological studies. Although genetic and environmental homogeneities exist, disease progressions can show large individual differences in animal models. In this study, we established an intact in vivo angiographic approach and explored vascular networks in the telencephalon of wild-type adult medaka using the spectral-domain optical coherence tomography. Our approach, which required neither surgical operations nor labeling agents, allowed to visualize blood vessels in medaka telencephala as small as about 8 µm, that is, almost the size of the blood cells of medaka. Besides, we could show the three-dimensional microvascular distribution in the medaka telencephalon. Therefore, the intact in vivo imaging via optical coherence tomography can be used to perform follow-up studies on cerebrovascular alterations in metabolic syndrome and their associations with neurodegenerative disease models in medaka.

Reduction of elastin in the skin causes various skin diseases as well as wrinkles and sagging with aging. Sialidase is a hydrolase that cleaves a sialic acid residue from sialoglycoconjugate. Cleavage of sialic acid from microfibrils by the sialidase isozyme Neu1 facilitates elastic fiber assembly. In the present study, we showed that a lower layer of the dermis and muscle showed relatively intense sialidase activity. The sialidase activity in the skin decreased with aging. Choline and geranate (CAGE), one of the ionic liquids, can deliver the sialidase subcutaneously while maintaining the enzymatic activity. The elastin level in the dermis was increased by applying sialidase from Arthrobacter ureafaciens (AUSA) with CAGE on the skin for 5 days in rats and senescence-accelerated mice prone 1 and 8. Sialidase activity in the dermis was considered to be mainly due to Neu2 based on the expression level of sialidase isozyme mRNA. Transdermal administration of Neu2 with CAGE also increased the level of elastin in the dermis. Therefore, not only Neu1 but also Neu2 would be involved in elastic fiber assembly. Transdermal administration of sialidase is expected to be useful for improvement of wrinkles and skin disorders due to the loss of elastic fibers.

To determine the risk of mask-associated dry eye (MADE), we investigated the fluorescein tear break-up time (FBUT), ocular surface temperature and blood flow, along with corneal sensitivity, in mask wearers. We enrolled 60 mask wearers (mean age, 27.1 ± 5.2 years) and then measured FBUT, corneal temperature and conjunctival blood flow without wearing masks (no mask), with masks, and with taped masks. We defined MADE as the condition in which dry eye symptoms appeared and the FBUT with mask was less than 5 s. The FBUT with a mask was significantly shorter compared to the no mask and taped mask groups (P < 0.01 and P < 0.05). The corneal temperature difference and conjunctival blood flow difference were significantly higher after wearing a mask than after wearing a taped mask (P < 0.01). Of the 60 subjects, 13 were diagnosed with MADE. Pain sensitivity and the Ocular Surface Disease Index (P < 0.05 and P < 0.01) were significantly higher in the MADE group, with the FBUT without masks (P < 0.05) significantly shorter than in the non-MADE group. MADE may be associated with corneal hypersensitivity. Wearing masks decreased FBUT and increased ocular surface temperature and blood flow. Taping the top edge of masks prevented these changes. Fitting masks properly may reduce MADE risk.

We present evidence for the decomposition and oxidation of amino acids in aqueous solution following irradiation with a nonequilibrium plasma jet. Of 15 amino acids tested in cell culture medium, plasma irradiation induced a marked chemical change in methionine and tryptophan due to the effective production of reactive oxygen species by plasma-water interaction. We also report that plasma-treated methionine and tryptophan aqueous solutions can kill cancer cells, greatly decreasing the viability of human endometrial carcinoma (HEC-1) cancer cells due to the presence of decomposition or oxidation products generated from the amino acid. Plasma-treated methionine and tryptophan aqueous solutions also induced an anti-cancer effect on cancer-initiating cells.

We investigated the effects of extracts of Benifuuki (a tea cultivar that contains methylated catechins such as epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3"Me)) in mice fed a high-fat/high-sucrose (HF/HS) diet. This tea cultivar was then compared with an extract of Yabukita (a popular tea cultivar that lacks methylated catechins). For 6 weeks, C57BL/6J mice were fed either HF/HS diet with or without tea extracts from tea cultivars, which contained almost identical ingredients except for methylated catechins (i.e., Yabukita (0.2% and 1%) or Benifuuki (0.2% and 1%) extract powders). Supplementation with Benifuuki 0.2% markedly lowered plasma levels of TG and NEFAs compared with mice supplemented with Yabukita 0.2%. The diet containing Benifuuki 1% decreased adipose tissue weights, liver TG, and expression of lipogenic genes in the liver. These results suggested that Benifuuki had much greater lipid-lowering effects than Yabukita. Taken together, these data suggest that methylated catechins direct the strong lipid-lowering activity of Benifuuki.

Breast cancer is the most common cancer type among women worldwide. The majority of breast cancer expresses estrogen receptor (ER) and endocrine therapy is a standard treatment of ER-positive breast cancer. However, development of the therapy resistance is still a major challenge and thus new therapeutic approaches are needed. Here we show that an RNA-binding protein, PSPC1, play a crucial role in ER-positive breast cancer growth through post-transcriptional gene regulation. We showed that siRNA-mediated PSPC1 silencing suppressed the proliferation of ER-positive breast cancer cells. Strong immunoreactivity (IR) of PSPC1 was correlated with poor prognosis for ER-positive breast cancer patients. Using immunoprecipitation, RNA-immunoprecipitation (RIP) and quantitative PCR (qPCR) experiments, we showed that PSPC1 interacted with PSF and was involved in post-transcriptional regulation of PSF target genes, ESR1 and SCFD2. Strong SCFD2 IR was correlated with poor prognosis for ER-positive breast cancer patients and combinations of PSPC1, PSF, and SCFD2 IRs were potent prognostic factors. Moreover, we identified DDIAS and MYBL1 as SCFD2 downstream target genes using microarray analysis, and finally showed that SCFD2 silencing suppressed tamoxifen-resistant breast tumor growth in vivo. These results indicated that PSPC1 and SCFD2 axis could be a promising target in the clinical management of the disease.