[Current status of breast cancer undetected by mammography in population-based breast cancer screening and associated factors].

Objective　This study aimed to promote the application of mammography (MMG) screening without performing a clinical breast examination (CBE). It examined population-based screening data, including history taking findings, to elucidate the status of breast cancer cases, detected solely by CBE, and to reveal the factors associated with breast cancer. Through this, it explored alternative methods for evaluating breast cancer cases, undetected by MMG, when CBE is omitted.Methods　The linked anonymized data from women, who underwent breast cancer screening in 2014, 2016, or 2017, were prepared. The data were obtained from the Nishinomiya City database. Breast cancer, undetected by MMG, were defined as breast cancer cases diagnosed by close examination based on CBE only (MMG findings were category 2 or lower, with no abnormalities). To assess the quality of breast cancer screening, process indices were calculated for the overall population, and for patients indicated for close examination based on CBE but not MMG. The association of breast cancer with each factor was statistically analyzed (χ2 test, etc.).Results　In total, 13,504 women underwent breast cancer screening. Close examination was required in 1,247 women (9.2%). Breast cancer was diagnosed in 44 women (3.5%), including four, who had breast cancer undetected by MMG. All of the process indices satisfied the acceptable values. Three of the four women with breast cancer, undetected by MMG, noticed a lump. Breast cancer was significantly associated with "subjective symptoms". The presence of a "lump" and "nipple discharge" were significantly more common in breast cancer patients.Conclusion　Three of the four breast cancer cases, undetected by MMG screening, had a subjective symptom (lump). There was a significant association between subjective symptoms (lump and nipple discharge) and breast cancer. To avoid missing breast cancer, undetected by MMG alone, these symptoms should be especially evaluated in women to compensate for the omission of a CBE. This can be achieved by encouraging women experiencing symptoms to seek consult, performing a thorough history-taking and observation, engaging in multidisciplinary collaboration (communication from medical staff to physicians), and promoting breast awareness.

Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells.

Gut commensal microbes shape the mucosal immune system by regulating the differentiation and expansion of several types of T cell. Clostridia, a dominant class of commensal microbe, can induce colonic regulatory T (Treg) cells, which have a central role in the suppression of inflammatory and allergic responses. However, the molecular mechanisms by which commensal microbes induce colonic Treg cells have been unclear. Here we show that a large bowel microbial fermentation product, butyrate, induces the differentiation of colonic Treg cells in mice. A comparative NMR-based metabolome analysis suggests that the luminal concentrations of short-chain fatty acids positively correlates with the number of Treg cells in the colon. Among short-chain fatty acids, butyrate induced the differentiation of Treg cells in vitro and in vivo, and ameliorated the development of colitis induced by adoptive transfer of CD4(+) CD45RB(hi) T cells in Rag1(-/-) mice. Treatment of naive T cells under the Treg-cell-polarizing conditions with butyrate enhanced histone H3 acetylation in the promoter and conserved non-coding sequence regions of the Foxp3 locus, suggesting a possible mechanism for how microbial-derived butyrate regulates the differentiation of Treg cells. Our findings provide new insight into the mechanisms by which host-microbe interactions establish immunological homeostasis in the gut.

Canagliflozin reduces plasma uremic toxins and alters the intestinal microbiota composition in a chronic kidney disease mouse model.

Accumulation of uremic toxins, which exert deleterious effects in chronic kidney disease, is influenced by the intestinal environment; the microbiota contributes to the production of representative uremic toxins, including p-cresyl sulfate and indoxyl sulfate. Canagliflozin is a sodium-glucose cotransporter (SGLT) 2 inhibitor, and it also exerts a modest inhibitory effect on SGLT1. The inhibition of intestinal SGLT1 can influence the gastrointestinal environment. We examined the effect of canagliflozin on the accumulation of uremic toxins in chronic kidney disease using adenine-induced renal failure mice. Two-week canagliflozin (10 mg/kg po) treatment did not influence the impaired renal function; however, it significantly reduced the plasma levels of p-cresyl sulfate and indoxyl sulfate in renal failure mice (a 75% and 26% reduction, respectively, compared with the vehicle group). Additionally, canagliflozin significantly increased cecal short-chain fatty acids in the mice, suggesting the promotion of bacterial carbohydrate fermentation in the intestine. Analysis of the cecal microbiota showed that canagliflozin significantly altered microbiota composition in the renal failure mice. These results indicate that canagliflozin exerts intestinal effects that reduce the accumulation of uremic toxins including p-cresyl sulfate. Reduction of accumulated uremic toxins by canagliflozin could provide a potential therapeutic option in chronic kidney disease.

Evaluation of the impact of gut microbiota on uremic solute accumulation by a CE-TOFMS-based metabolomics approach.

Gut microbiota is involved in the metabolism of uremic solutes. However, the precise influence of microbiota to the retention of uremic solutes in CKD is obscure. To clarify this, we compared adenine-induced renal failure and control mice under germ-free or specific pathogen-free (SPF) conditions, examining the metabolite profiles of plasma, feces, and urine using a capillary electrophoresis time-of-flight mass spectrometry-based approach. Mice with renal failure under germ-free conditions demonstrated significant changes in plasma metabolites. Among 183 detected solutes, plasma levels of 11 solutes, including major uremic toxins, were significantly lower in germ-free mice than in SPF mice with renal failure. These 11 solutes were considered microbiota-derived uremic solutes and included indoxyl sulfate, p-cresyl sulfate, phenyl sulfate, cholate, hippurate, dimethylglycine, γ-guanidinobutyrate, glutarate, 2-hydroxypentanoate, trimethylamine N-oxide, and phenaceturate. Metabolome profiling showed that these solutes were classified into three groups depending on their origins: completely derived from microbiota (indoxyl sulfate, p-cresyl sulfate), derived from both host and microbiota (dimethylglycine), and derived from both microbiota and dietary components (trimethylamine N-oxide). Additionally, germ-free renal failure conditions resulted in the disappearance of colonic short-chain fatty acids, decreased utilization of intestinal amino acids, and more severe renal damage compared with SPF mice with renal failure. Microbiota-derived short-chain fatty acids and efficient amino acid utilization may have a renoprotective effect, and loss of these factors may exacerbate renal damage in germ-free mice with renal failure. Thus, microbiota contributes substantially to the production of harmful uremic solutes, but conversely, growth without microbiota has harmful effects on CKD progression.

Comparison of CAGE and RNA-seq transcriptome profiling using clonally amplified and single-molecule next-generation sequencing.

CAGE (cap analysis gene expression) and RNA-seq are two major technologies used to identify transcript abundances as well as structures. They measure expression by sequencing from either the 5' end of capped molecules (CAGE) or tags randomly distributed along the length of a transcript (RNA-seq). Library protocols for clonally amplified (Illumina, SOLiD, 454 Life Sciences [Roche], Ion Torrent), second-generation sequencing platforms typically employ PCR preamplification prior to clonal amplification, while third-generation, single-molecule sequencers can sequence unamplified libraries. Although these transcriptome profiling platforms have been demonstrated to be individually reproducible, no systematic comparison has been carried out between them. Here we compare CAGE, using both second- and third-generation sequencers, and RNA-seq, using a second-generation sequencer based on a panel of RNA mixtures from two human cell lines to examine power in the discrimination of biological states, detection of differentially expressed genes, linearity of measurements, and quantification reproducibility. We found that the quantified levels of gene expression are largely comparable across platforms and conclude that CAGE and RNA-seq are complementary technologies that can be used to improve incomplete gene models. We also found systematic bias in the second- and third-generation platforms, which is likely due to steps such as linker ligation, cleavage by restriction enzymes, and PCR amplification. This study provides a perspective on the performance of these platforms, which will be a baseline in the design of further experiments to tackle complex transcriptomes uncovered in a wide range of cell types.

Alteration of the Intestinal Environment by Lubiprostone Is Associated with Amelioration of Adenine-Induced CKD.

The accumulation of uremic toxins is involved in the progression of CKD. Various uremic toxins are derived from gut microbiota, and an imbalance of gut microbiota or dysbiosis is related to renal failure. However, the pathophysiologic mechanisms underlying the relationship between the gut microbiota and renal failure are still obscure. Using an adenine-induced renal failure mouse model, we evaluated the effects of the ClC-2 chloride channel activator lubiprostone (commonly used for the treatment of constipation) on CKD. Oral administration of lubiprostone (500 µg/kg per day) changed the fecal and intestinal properties in mice with renal failure. Additionally, lubiprostone treatment reduced the elevated BUN and protected against tubulointerstitial damage, renal fibrosis, and inflammation. Gut microbiome analysis of 16S rRNA genes in the renal failure mice showed that lubiprostone treatment altered their microbial composition, especially the recovery of the levels of the Lactobacillaceae family and Prevotella genus, which were significantly reduced in the renal failure mice. Furthermore, capillary electrophoresis-mass spectrometry-based metabolome analysis showed that lubiprostone treatment decreased the plasma level of uremic toxins, such as indoxyl sulfate and hippurate, which are derived from gut microbiota, and a more recently discovered uremic toxin, trans-aconitate. These results suggest that lubiprostone ameliorates the progression of CKD and the accumulation of uremic toxins by improving the gut microbiota and intestinal environment.

Gut microbiome-derived phenyl sulfate contributes to albuminuria in diabetic kidney disease.

Diabetic kidney disease is a major cause of renal failure that urgently necessitates a breakthrough in disease management. Here we show using untargeted metabolomics that levels of phenyl sulfate, a gut microbiota-derived metabolite, increase with the progression of diabetes in rats overexpressing human uremic toxin transporter SLCO4C1 in the kidney, and are decreased in rats with limited proteinuria. In experimental models of diabetes, phenyl sulfate administration induces albuminuria and podocyte damage. In a diabetic patient cohort, phenyl sulfate levels significantly correlate with basal and predicted 2-year progression of albuminuria in patients with microalbuminuria. Inhibition of tyrosine phenol-lyase, a bacterial enzyme responsible for the synthesis of phenol from dietary tyrosine before it is metabolized into phenyl sulfate in the liver, reduces albuminuria in diabetic mice. Together, our results suggest that phenyl sulfate contributes to albuminuria and could be used as a disease marker and future therapeutic target in diabetic kidney disease.

Comparison of Chromogenic Selective Media for the Detection of Cronobacter spp. (Enterobacter sakazakii).

　The four types of chromogenic selective media that are commercially available in Japan were compared for establishing a Japanese standard method for detecting Cronobacter spp. based on ISO/TS 22964:2006. When assessed using 9 standard Cronobacter spp. strains and 29 non-Cronobacter strains, Enterobacter sakazakii isolation agar, ChromocultTM Enterobacter sakazakii agar, CHROMagarTM E. sakazakii, and XM-sakazakii agar demonstrated excellent inclusivity and exclusivity. Using the ISO/TS 22964:2006 method, the recovered numbers of 38 Cronobacter spp. strains, including 29 C. sakazakii isolates obtained from each medium, were equivalent, indicating that there was no significant difference (p > 0.05) among the four types of chromogenic selective media. Thus, we demonstrated that these four chromogenic selective media are suitable alternatives when using the standard method for detecting Cronobacter spp. in Japan, based on the ISO/TS 22964:2006.

Eastern blotting and immunoaffinity concentration using monoclonal antibody for ginseng saponins in the field of traditional chinese medicines.

Ginsenosides separated by silica gel TLC blotted to a PVDF membrane that was treated with a NaIO4 solution followed by bovine serum albumin (BSA) resulted in a ginsenoside-BSA conjugate on a PVDF membrane. The blotted spots were stained by anti-ginsenoside Rb1 (G-Rb1) and -Rg1 (G-Rg1) monoclonal antibodies (MAbs). The newly established immunostaining method, Eastern blotting, was applied for the determination of ginsenosides possessing protopanaxadiol and/or protopanaxatriol in the traditional Chinese medicine (TCM). This method developed a new way to separate the ginsenoside molecule into two functional parts using a simple and well-known chemical reaction. The sugar parts were oxidized by NaIO4 to give dialdehydes, which reacted with amino groups of the protein and covalently bound to the adsorbent PVDF membrane. The MAb bound to the aglycon part of the ginsenoside molecule for immunostaining. Double staining of Eastern blotting for ginsenosides using anti-G-Rb1 and -Rg1 MAbs promoted complete identification of ginsenosides in Panax species. The immunoaffinity concentration of G-Rb1 was deteremined by immunoaffinity column conjugated with anti-G-Rb1 MAb leading to the knock-out extract, which will be useful for the pharmacological investigation. To concentrate and determine G-Rb1 in P. japonicus, the crude extract of P. japonicus was fractionated by immunoaffinity column conjugated with anti-G-Rb1 MAb. Two ginsenosides, chikusetsusaponins III and IV having protopanaxadiol as an aglycon, were identified by Eastern blotting, although it was expected that G-Rb1 might be a component of P. japonicus by enzyme-linked immunosorbent assay (ELISA) analysis.

Cronobacter spp. in commercially available dried food in Japan.

A total of 140 samples of dried food sold in Japan were surveyed and tested for the presence of viable bacteria, distribution of coliform bacteria, and contamination with Cronobacter spp. The samples were purchased from retail stores in Tokyo and Kanagawa Prefecture. Out of the 140 samples tested, viable bacteria were found in 135 samples and coliform bacteria were found in 23 samples. Qualitative and quantitative testing revealed the presence of Cronobacter spp. in 35 (25.0%) and 11 samples (7.9%), respectively. The most commonly found Cronobacter species were C. sakazakii, with the next most common, in order, being C. muytjensii and C. turicensis. The actual numbers of Cronobacter species in the tested dried foods were low, but the widespread contamination particularly in dried herbs and vegetables was confirmed.

Preoperative epidural fentanyl reduces postoperative pain after upper abdominal surgery.

Forty patients, American Society of Anesthesiology (ASA) physical status 1-2, undergoing subtotal gastrectomy were enrolled in this study. The patients were allocated to two groups with or (group P) and without (group C) preoperative epidural fentanyl 100 microg. Postoperatively, all patients received continuous infusion of the study solution, containing fentanyl 30 microg x ml(-1) and 2 mg/ml bupivacaine, at a rate of 0.7 ml x h(-1) for 72 h. The scores on the Prince Henry Hospital self-assessed pain scale (PHPS) were recorded at 0, 4, 12, 24, 48, and 72 h after the surgery. We compared the total rescue doses of analgesics during each period of 24 h until 72 h postoperatively. Although the total rescue doses of analgesics were not different between the groups, the median PHPS score was lower in group P than in group C, except at 0 h after the surgery. Preoperative epidural fentanyl 100 microg may increase the analgesic potency of postoperative epidural low-dose infusion of bupivacaine with fentanyl.

Identification and differentiation of Panax species using ELISA, RAPD and eastern blotting.

Immunochemical and genetic methods have been developed in order to distinguish Panax spp. With the aim of establishing immunochemical methods, two hybridomas (3H4 and 5H8), each secreting a monoclonal antibody (MAb) against proteins of Panax ginseng, were prepared by fusing splenocytes immunized with two kinds of ginseng water-soluble fractions and a hypoxanthine-thymidine-aminopterin-sensitive mouse myeloma cell line, P3-X63-Ag8-U1. MAb 3H4 cross-reacted with four Panax spp., whereas the MAb 5H8 cross-reacted with P. ginseng in the enzyme-linked immunosorbent assay (ELISA). ELISA and western blotting methods using a ginseng water-soluble fraction as the solid-phase antigen were developed for the unambiguous authentication of P. ginseng. A combination of random amplified polymorphic DNA (RAPD) and eastern blotting analyses using anti-ginsenoside Rb1 and Rgl monoclonal antibodies was used for the identification of P. notoginseng, P. quinquefolius and P. japonicus. RAPD can be used to differentiate the species of Panax from each other. An important parameter used for differentiating P. notoginseng is the absence of ginsenoside Rc in the extract of P. notoginseng with eastern blotting. The combination of these methods enabled a reliable identification of Panax spp.

Chromatographic resolution of glucosidic compounds, ginsenosides on polyethersulphone membrane, and its application to the quantitative immunoassay for ginseng saponins.

A method has been devised for the chromatographic resolution of glucosidic compounds, ginseng saponins, on polyethersulphone (PES) membrane. The method results in good resolution and quantitative immunoassay for ginsenoside Rb1 (G-Rb1), G-Rc, and G-Rd in crude extracts of various ginsengs. The newly established method is simpler and applies for quantitative analysis. Ginsenosides developed by acetonitrile-water-acetic acid solvent system on a PES membrane were directly treated with a NaIO4 solution followed by bovine serum albumin (BSA), resulting in a ginsenoside-BSA conjugate on a PES membrane. Anti-G-Rb1 monoclonal antibody (MAb) was bound, and then a second antibody labeled with peroxidase directed against the first antibody. Finally a substrate reacted to the enzyme and gave staining. The stained membrane was scanned, and spots were analyzed quantitatively using NIH Image software. At least 62.5 ng of G-Rb1, G-Rc, and G-Rd were clearly detectable individually. Three ginsenosides can be analyzed quantitatively between 0.125 and 2.0 microg.

Isolation of ginsenoside Rb1 from Kalopanax pictus by eastern blotting using anti-ginsenoside Rb1 monoclonal antibody.

Araliaceous species containing ginsenosides, the major active component in Panax species, were surveyed using ELISA and eastern blotting with anti-ginsenoside Rb1 monoclonal antibody. Immunoassay-guided fractionation of the methanol-soluble extracts of Araliaceous species led to the isolation of a known compound in the bark of Kalopanax pictus Nakai. The known compound was identified as ginsenoside Rb1 by spectroscopic methods and by comparison with an authentic sample. The result provided evidence that a combination of two immunoassays using monoclonal antibodies against ginsenosides is a powerful means of surveying new ginsenoside resources.

A one-step immunochromatographic assay for detecting ginsenosides Rb1 and Rg1.

An immunochromatographic strip test has been developed for detecting ginsenosides Rb1 (G-Rb1) and Rg1 (G-Rg1). This qualitative assay system is useful as a rapid screening method for detecting G-Rb1 and G-Rg1 in plants and plant preparations. Our assay is a competitive immunoassay that uses anti-G-Rb1 and anti-G-Rg1 monoclonal antibodies (MAbs) and a detection reagent that contains colloidal gold particles coated with anti-G-Rb1 and anti-G-Rg1 MAbs. Detection limits are 2 microg mL(-1) for both G-Rb1 and G-Rg1.