Site-specific glycosylation and single amino acid substitution dramatically reduced the immunogenicity of ß-lactoglobulin.

To reduce the immunogenicity of ß-lactoglobulin (BLG), we prepared recombinant BLG which has both site-specific glycosylation and single amino acid substitution (D28N/P126A), and expressed it in the methylotrophic yeast Pichia pastoris by fusion of the cDNA to the sequence coding for the α-factor signal peptide from Saccharomyces cerevisiae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the D28N/P126A was conjugated with a ∼4 kDa high-mannose chain. D28N/P126A retained ∼61% of the retinol-binding activity of BLG. Structural analyses by circular dichroism (CD) spectra, intrinsic fluorescence, and Enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies indicated that the surface structure of BLG was slightly changed by using protein engineering techniques, but D28N/P126A was covered by high-mannose chains and substituted amino acid without substantial disruption of native conformation. Antibody responses to the D28N/P126A considerably reduced in C57BL/6 mice. We conclude that inducing both site-specific glycosylation and single amino acid substitution simultaneously is an effective method to reduce the immunogenicity of BLG.

[Epidemiological investigation of colonic diverticulosis detected by computed tomography colonography].

Among 586 patients who underwent computed tomography colonography (CTC) from May 2012 to September 2017, 333 were diagnosed with colonic diverticulosis. The incidence of colonic diverticulosis increases with age. Despite a high frequency of ascending colonic diverticulosis, multiple diverticulosis (>10 in a colonic segment) were the most frequent in the sigmoid colon. In previous studies, the frequency of detection of colonic diverticulosis by CTC was higher than that by colonoscopy and barium enema. In addition, using CTC, the detection rate of colonic diverticulosis has been recently increasing, suggesting that CTC is the most sensitive procedure for detecting colonic diverticulosis.

Dammarane-type triterpene extracts of Panax notoginseng root ameliorates hyperglycemia and insulin sensitivity by enhancing glucose uptake in skeletal muscle.

Skeletal muscle is an important organ for controlling the development of type 2 diabetes. We discovered Panax notoginseng roots as a candidate to improve hyperglycemia through in vitro muscle cells screening test. Saponins are considered as the active ingredients of ginseng. However, in the body, saponins are converted to dammarane-type triterpenes, which may account for the anti-hyperglycemic activity. We developed a method for producing a dammarane-type triterpene extract (DTE) from Panax notoginseng roots and investigated the extract's potential anti-hyperglycemic activity. We found that DTE had stronger suppressive activity on blood glucose levels than the saponin extract (SE) did in KK-Ay mice. Additionally, DTE improved oral glucose tolerance, insulin sensitivity, glucose uptake, and Akt phosphorylation in skeletal muscle. These results suggest that DTE is a promising agent for controlling hyperglycemia by enhancing glucose uptake in skeletal muscle.

Effects of Food-Derived Collagen Peptides on the Expression of Keratin and Keratin-Associated Protein Genes in the Mouse Skin.

Oral ingestion of collagen peptides (CP) has long been suggested to exert beneficial effects on the skin, but the molecular events induced by CP on the skin remain unclear. Here, we investigated the effects of oral CP administration on gene expression in hairless mouse skin and of prolyl-hydroxyproline (Pro-Hyp), a collagen-derived dipeptide, on gene expression in a coculture of mouse skin keratinocytes and fibroblasts. Using microarray analysis, we found that oral administration of CP to hairless mice for 6 weeks induced increased expression of Krtap and Krt genes in the skin. Annotation analysis using DAVID revealed that a group of the up-regulated genes, Gprc5d, Sprr2a1, Krt27 and Krtap16-7, is associated with the development of the epidermis and the hair cycle. In addition, the presence of Pro-Hyp (200 µM) induced an increase in the expression of Krtap16-7, Krtap15, Krtap14 and Krtap8-2 in keratinocytes in coculture, partially resembling the in vivo result. The Pro-Hyp-induced up-regulation of these genes was not observed when keratinocytes were cultured without fibroblasts, suggesting that the presence of fibroblasts is essential for the effects of Pro-Hyp. Our study presents new insights into the effects of CP on the skin, which might link to the hair cycle.

The role of non-enhanced angiography in toe tip transfer with small diameter pedicle.

BACKGROUND: Toe tip transfer allows functional and esthetic reconstruction of the lost fingertip, but it is still uncommon because identification and dissection of donor and recipient veins can be challenging. Nonenhanced angiography (NEA) is a device that emits infrared light at a wavelength of 850 nm, which is exclusively absorbed by hemoglobin. The light penetrates the bones and other soft tissues, effectively visualizing veins in real time. The aim of this report is to present the experience on the preoperative use of nonenhanced angiography for visualization of donor and recipient veins in toe tip transfers in a series of patients. PATIENTS AND METHODS: Four cases of toe tip transfer and one case of free nail flap were performed for reconstruction of the tips of thumb and finger with preoperative examination using NEA. Patients' age ranged from 29 to 52 years old (average, 29.2 years old). Before the operation, the veins in the donor and recipient sites were marked using NEA, and the blood flow of the veins in the recipient site was confirmed. RESULTS: Pedicles in all transferred toe tips were less than 2 cm in length, with diameters smaller than 0.8 mm. The postoperative courses were uneventful, and all transferred toe tips survived completely, with satisfying functional and aesthetic results. CONCLUSIONS: NEA may facilitate venous dissection of the donor and the recipient sites, allowing safe and efficient toe tip transfer with a small pedicle.

Antigen presentation by small intestinal epithelial cells uniquely enhances IFN-γ secretion from CD4+ intestinal intraepithelial lymphocytes.

Small intestinal epithelial cells (sIECs) express major histocompatibility complex class II molecules even in a normal condition, and are known to function as antigen presenting cells (APCs) at least in vitro. These findings raised the possibility that sIECs play an important role in inducing immune responses against luminal antigens, especially those of intestinal intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). We herein showed that antigenic stimulation with sIECs induced markedly greater secretion of interferon-gamma (IFN-γ) by CD4(+) IELs, but not interleukin (IL)-4, IL-10 and IL-17 although the proliferative response was prominently lower than that with T cell-depleted splenic APCs. In contrast, no enhanced IFN-γ secretion by CD4(+) LPLs and primed splenic CD4(+) T cells was observed when stimulated with sIECs. Taken together, these results suggest that sIECs uniquely activate CD4(+) IELs and induce remarkable IFN-γ secretion upon antigenic stimulation in vivo.

Effects of oral administration of glucosylceramide on gene expression changes in hairless mouse skin: comparison of whole skin, epidermis, and dermis.

The beneficial effects of dietary glucosylceramide on the barrier function of the skin have been increasingly reported, but the entire mechanism has not been clarified. By DNA microarray, we investigated changes in gene expression in hairless mouse skin when a damage-inducing AD diet and a glucosylceramide diet (GluCer) were imposed. GluCer administration potentially suppressed the upregulation of six genes and the downregulation of four genes in the AD group. Examination of the epidermal and/or dermal expression of Npr3, Cyp17a1, Col1a1, S100a9, Sprr2f, Apol7a, Tppp, and Scd3 revealed responses of various parts of the skin to the diets. In normal hairless mice, GluCer administration induced an increase in the dermal expression of Cyp17a1 and the epidermal expression of Tppp, and a decrease in the epidermal expression of S100a9. Our results provide information on gene expression not only in whole skin but also in the epidermis and dermis that should prove useful in the search for the mechanisms underlying the effects of GluCer on damaged and normal skin.

Upregulation of CREB-mediated transcription enhances both short- and long-term memory.

Unraveling the mechanisms by which the molecular manipulation of genes of interest enhances cognitive function is important to establish genetic therapies for cognitive disorders. Although CREB is thought to positively regulate formation of long-term memory (LTM), gain-of-function effects of CREB remain poorly understood, especially at the behavioral level. To address this, we generated four lines of transgenic mice expressing dominant active CREB mutants (CREB-Y134F or CREB-DIEDML) in the forebrain that exhibited moderate upregulation of CREB activity. These transgenic lines improved not only LTM but also long-lasting long-term potentiation in the CA1 area in the hippocampus. However, we also observed enhanced short-term memory (STM) in contextual fear-conditioning and social recognition tasks. Enhanced LTM and STM could be dissociated behaviorally in these four lines of transgenic mice, suggesting that the underlying mechanism for enhanced STM and LTM are distinct. LTM enhancement seems to be attributable to the improvement of memory consolidation by the upregulation of CREB transcriptional activity, whereas higher basal levels of BDNF, a CREB target gene, predicted enhanced shorter-term memory. The importance of BDNF in STM was verified by microinfusing BDNF or BDNF inhibitors into the hippocampus of wild-type or transgenic mice. Additionally, increasing BDNF further enhanced LTM in one of the lines of transgenic mice that displayed a normal BDNF level but enhanced LTM, suggesting that upregulation of BDNF and CREB activity cooperatively enhances LTM formation. Our findings suggest that CREB positively regulates memory consolidation and affects memory performance by regulating BDNF expression.

Effect of taurine on mRNA expression of thioredoxin interacting protein in Caco-2 cells.

Taurine (2-aminoethanesulfonic acid), a sulfur-containing ß-amino acid, plays an important role in several essential biological processes; although, the underlying mechanisms for these regulatory functions remain to be elucidated, especially at the genetic level. We investigated the effects of taurine on the gene expression profile in Caco-2 cells using DNA microarray. Taurine increased the mRNA expression of thioredoxin interacting protein (TXNIP), which is involved in various metabolisms and diseases. ß-Alanine or γ-aminobutyric acid (GABA), which are structurally or functionally related to taurine, did not increase TXNIP mRNA expression. These suggest the expression of TXNIP mRNA is induced specifically by taurine. ß-Alanine is also known to be a substrate of taurine transporter (TAUT) and competitively inhibits taurine uptake. Inhibition of taurine uptake by ß-alanine eliminated the up-regulation of TXNIP, which suggests TAUT is involved in inducing TXNIP mRNA expression. The up-regulation of TXNIP mRNA expression by taurine was also observed at the protein level. Furthermore, taurine significantly increased TXNIP promoter activity. Our present study demonstrated the taurine-specific phenomenon of TXNIP up-regulation, which sheds light on the physiological function of taurine.

Decreased Frequency of Mental Workload-Induced Subjective Hot Flashes Through Gum Massage: An Open-Label, Self-Controlled Crossover Trial.

Objective: Hot flashes, a symptom of menopause, can decrease women's quality of life. Sympathetic nervous system activation has been identified as an important factor in the occurrence of hot flashes. Given that somatosensory stimulation of the oral cavity can affect autonomic nervous activity, we aimed to investigate the possibility that somatosensory stimulation of the gums (i.e., gum massage) could improve hot flashes. Materials and Methods: Nineteen women experiencing at least one hot flash per day were instructed to perform a gum massage on themselves before undertaking mental workload, using arithmetic task, and the frequency of hot flashes experienced during this task was measured. Changes in autonomic nervous activity were assessed based on heart rate variability. Results: Massage conditions promoted a significantly lower arithmetic task-induced hot flash frequency compared with nonmassage conditions (p < 0.05). During gum massage, the ratio between low and high frequency (LF/HF) values decreased significantly under massage conditions compared with nonmassage conditions (p < 0.01). During the arithmetic task, the gum massage-induced reduction in LF/HF, which changed from baseline, was significantly correlated with the gum massage-induced reduction in hot flash frequency. Conclusions: The results of this study indicate that gum massage can reduce the subjective frequency of hot flashes over a certain period under mental workload. Our study also indicates that gum massage can potentially decrease sympathetic nerve activity, which is known to be involved in the occurrence of hot flashes.Clinical Trial Registration number 328 (the institutional review board of Lion Corporation).

Modified BEST-J Score Model Predicts Bleeding after Endoscopic Submucosal Dissection with Fewer Factors.

This study constructed a simplified post-endoscopic submucosal dissection (ESD) prediction model with a prognostic nutritional index (PNI). A total of 449 patients who underwent gastric ESD was included, divided with a ratio of 2:1, and assigned to the model or validation cohort. A prediction model of post-ESD (modified BEST-J score) was constructed using the model cohort. The modified BEST-J score was evaluated by comparing its accuracy to the BEST-J score in the validation cohort. Within 4 weeks of ESD, melena, hematemesis, or a 2 g/dL or greater decrease in hemoglobin level that required esophagogastroduodenoscopy was defined as post-ESD bleeding. In the model cohort, 299 patients were enrolled and 25 (8.4%) had post-ESD bleeding. Independent risk factors for post-ESD bleeding were use of P2Y12RA, tumor size > 30 mm, location of lesion at lower one-third of the stomach, and PNI ≤ 47.9. Constructing the modified BEST-J score based on these variables, the sensitivity, specificity, and positive likelihood ratio were 73.9%, 78.1%, and 3.37. When comparing the modified BEST-J score to the BEST-J score in the validation cohort, no significant difference was observed by ROC-AUC (0.77 vs. 0.75, p = 0.81). Modified BEST-J score can predict post-ESD bleeding more simply, with the same accuracy as the BEST-J score.

Establishment of intestinal epithelial cell lines from adult mouse small and large intestinal crypts.

Small and large intestinal epithelial cell (IEC) lines were established from adult murine intestinal crypts. Both established small and large IECs line (named aMoS7 and aMoC1 respectively) expressed epithelial markers. Similarly to IECs isolated from adult mouse intestines, the expression of major histocompatibility complex (MHC) class II molecules was induced by interferon-γ-treatment in both established cell lines. This expression of MHC class II molecules was higher in small intestinal aMoS7 cells than in large intestinal aMoC1 cells. Treatment with lipopolysaccharide and with ligands of Toll-like receptors 1, 2, 3, and 7 induced secretion of interleukin-6 from both adult IEC lines. These results suggest that the aMoS7 and aMoC1 cell lines can serve as useful tools in analyzing the immunological functions of IECs, especially in studying the IEC response to microbial components and its antigen presenting ability.

Effects of chondroitin sulfate and its oligosaccharides on toll-like receptor-mediated IL-6 secretion by macrophage-like J774.1 cells.

The effects of two types of chondroitin sulphate (CS), CS-A and CS-C, their oligosaccharides (oligo-CSs), and disaccharides (Di-CSs) on toll-like receptor (TLR)-mediated secretion of interleukin (IL)-6 were compared using macrophage-like cell line J774.1. IL-6 secretion in the J774.1 cells was markedly increased by Pam3CS4, LPS, and CpG, the ligands to TLR1/2, 4, and 9 respectively. Among these three ligands, CpG-induced IL-6 was most clearly suppressed by CSs and their digests. Suppression of IL-6 secretion by smaller sized CS-A was stronger than that by intact CS-A, whereas no such size-dependent suppression was apparent for CS-C. Di-4S, the disaccharide unit of the CS-A digest, also showed much stronger suppression than Di-6S, the disaccharide unit of the CS-C digest, and the non-sulfated disaccharide unit, Di-0S. The suppressing activity of oligo-CSs, particularly Di-CSs, against TLR-mediated inflammation was dependent on the CS structure, including the sulfation site.

The inhibitory effects of toothpaste and mouthwash ingredients on the interaction between the SARS-CoV-2 spike protein and ACE2, and the protease activity of TMPRSS2 in vitro.

SARS-CoV-2 enters host cells when the viral spike protein is cleaved by transmembrane protease serine 2 (TMPRSS2) after binding to the host angiotensin-converting enzyme 2 (ACE2). Since ACE2 and TMPRSS2 are expressed in the tongue and gingival mucosa, the oral cavity is a potential entry point for SARS-CoV-2. This study evaluated the inhibitory effects of general ingredients of toothpastes and mouthwashes on the spike protein-ACE2 interaction and the TMPRSS2 protease activity using an in vitro assay. Both assays detected inhibitory effects of sodium tetradecene sulfonate, sodium N-lauroyl-N-methyltaurate, sodium N-lauroylsarcosinate, sodium dodecyl sulfate, and copper gluconate. Molecular docking simulations suggested that these ingredients could bind to inhibitor-binding site of ACE2. Furthermore, tranexamic acid exerted inhibitory effects on TMPRSS2 protease activity. Our findings suggest that these toothpaste and mouthwash ingredients could help prevent SARS-CoV-2 infection.

Disaccharide derived from chondroitin sulfate A suppressed CpG-induced IL-6 secretion in macrophage-like J774.1 cells.

Interleukin (IL)-6 secretion from macrophage cells is known to be induced by toll-like receptor (TLR) 9 ligands, CpG (microbial DNA sequences containing unmethylated CpG dinucleotides). We have found, using macrophage-like J774.1 cells, that this induction was dramatically suppressed by a disaccharide derived from chondroitin sulfate A (Di-4S), but not by chondroitin sulfate A (CS-A) itself. The suppression of IL-6 secretion by Di-4S occurred at protein and mRNA expression levels. Di-4S inhibited the degradation of interleukin-1 receptor-associated kinase 1 (IRAK1) in the signaling pathway mediated by myeloid differentiation primary response gene (88) (MyD88) when stimulated by TLR9 activation. In addition to suppressing IRAK1 activation, interference with CpG-TLR9 interaction by Di-4S is also suggested to be one of the mechanisms. Oligosaccharides derived from chondroitin sulfates would be effective suppressing agents for the TLR9-mediated inflammation reaction.

Obstructive cholangitis by mucus from an intraductal papillary mucinous neoplasm with pancreatobiliary fistula treated by endoscopic septotomy and direct peroral cholangioscopy: a case report.

Intraductal papillary mucinous neoplasms (IPMNs) occasionally form a fistula to adjacent organs, resulting in obstructive jaundice and cholangitis due to mucus obstruction. Although some procedures such as endoscopic nasobiliary drainage are attempted, they often do not work adequately because of high mucus viscosity. Herein, we report the case of an 87-year-old man with obstructive cholangitis treated by endoscopic septotomy and mucus suction with direct peroral cholangioscopy using conventional endoscopy. The patient incidentally showed a branched-type IPMN in the pancreatic head on computed tomography (CT) approximately 10-years ago. Although the patient's tumor had grown slowly and he occasionally developed cholangitis, he did not want surgery. He was admitted to our hospital because of cholangitis by mucus obstruction with a PB fistula. Endoscopic retrograde cholangiopancreatography (ERCP) and septotomy were performed. Septotomy made the duodenal papilla a large orifice, thereby facilitating spontaneous drainage of mucus. In addition, conventional endoscopy with a large working channel enabled direct access into the orifice and smooth mucus suction, thereby alleviating his cholangitis. In conclusion, septotomy and direct peroral cholangioscopy using conventional endoscopy could be useful to control biliary tract infection and obstructive jaundice due to mucus obstruction from an IPMNs with PB fistula.

Association of Clinical Features with Human Leukocyte Antigen in Japanese Patients with Ulcerative Colitis.

BACKGROUND: The human leukocyte antigen (HLA) region has been found to be involved in the pathogenesis of inflammatory bowel disease (IBD), which is classified into ulcerative colitis (UC) and Crohn's disease (CD), by genome-wide association studies. The aim of this study was to confirm whether HLA-alleles confer susceptibility to UC and to determine whether HLA-allel1es are associated with the clinical phenotypes in Japanese patients with UC. METHODS: In this study, HLA typing was performed by PCR-sequence-specific oligonucleotides (PCR-SSO) to confirm the correlation between UC and HLA alleles (for HLA-A, B, DRB1) in 45 Japanese UC patients. In addition, whether the HLA alleles are related to patient and clinical background characteristics was examined. RESULTS: Overall, 62.2%, and 66.7% of the 45 UC patients had HLA-B*52 and HLA-DRB1*15, respectively. These allele frequencies were significantly higher than in previously reported Japanese control persons (P < 0.0001). The frequencies of extraintestinal manifestations [odds ratio (OR) = 0.12, P = 0.039] and a history of colectomy (OR = 0.18, P = 0.046) were lower in HLA-B*52-positive UC patients than in HLA-B*52 negative UC patients. The white blood cell (WBC) count was significantly higher in HLA-DRB1*15-positive patients (9430 ± 4592/µL) than in HLA-DRB1*15-negative patients (6729 ± 2160/µL). Thus, HLA-B*52 and DRB1*15 appear to be associated with disease features and severity in Japanese UC patients. CONCLUSION: These results indicate that HLA-B*52 and DRB1*15 are not only associated with overall UC susceptibility, but also with the clinical phenotypes in Japanese patients.

Expression of doublecortin and CaM kinase-like-1 protein in serrated neoplasia of the colorectum.

The adenoma-carcinoma sequence (ACS) and the serrated pathway are two distinct developmental routes leading to the formation of colorectal carcinoma. Recently, the doublecortin and CaM kinase-like-1 protein (DCLK1) has been reported to serve as an intestinal cancer stem cell marker and has been demonstrated to be overexpressed through the ACS; however, there is a lack of reports on the role of DCLK1 in the serrated pathway. To clarify the correlation between DCLK1 protein expression and clinicopathological characteristics of the serrated tumorigenic pathway, the present study used immunohistochemistry to examine the expression of DCLK1 in endoscopically resected samples of 62 serrated polyps [20 hyperplastic polyps (HPs), 16 traditional serrated adenomas (TSAs) and 26 sessile serrated adenoma-polyps (SSA/Ps)], as well as 20 non-serrated adenomas, 20 carcinoma in adenomas (CIAs) and 18 early pure colorectal carcinomas without any adenoma component (EPCs). Based on immunostaining score, high DCLK1 expression was detected in 20.0% of HPs (23.1% of microvesicular HPs and 14.3% of goblet cell HPs), 37.5% of TSAs, 7.7% of SSA/Ps, 80.0% of non-serrated adenomas, 75.0% of CIAs and 50.0% of EPCs. Negative or low DCLK1 expression was frequently observed in TSAs (P<0.005), SSA/Ps (P<0.00001) and EPCs (P<0.04) compared with non-serrated adenomas and CIAs. In addition, negative or low DCLK1 expression was significantly more frequent in SSA/Ps (92.3%) compared with TSAs (62.5%; P<0.05). Thus, the expression pattern of DCLK1 between the serrated pathway and ACS differed, indicating that DCLK1 expression may perform a secondary role in serrated tumorigenesis. In addition, the data indicates that EPCs may contain tumors derived from the serrated pathway as well as the ACS.

Interactions between αCaMKII and calmodulin in living cells: conformational changes arising from CaM-dependent and -independent relationships.

BACKGROUND: αCaMKII plays central and essential roles in long-term potentiation (LTP), learning and memory. αCaMKII is activated via binding with Ca²âº/CaM in response to elevated Ca²âº concentration. Furthermore, prolonged increase in Ca²âº concentration leads to the auto-phosphorylation of αCaMKII at T286, maintaining the activation of αCaMKII even after Ca²âº/CaM dissociation. Importantly, the active form of αCaMKII is thought to exhibit conformational change. In order to elucidate the relationships between the interaction of αCaMKII with CaM and the conformational change of αCaMKII, we generated molecular probes (YFP-αCaMKII with CFP-CaM and YFP-αCaMKII-CFP) and performed time-lapse imaging of the interaction with CaM and the conformational change, respectively, in living cells using FRET. RESULTS: The interaction of YFP-αCaMKII with CFP-CaM and the conformational change of YFP-αCaMKII-CFP were induced simultaneously in response to increased concentrations of Ca²âº. Consistent with previous predictions, high levels of Ca²âº signaling maintained the conformational change of YFP-αCaMKII-CFP at the time when CFP-CaM was released from YFP-αCaMKII. These observations indicated the transfer of αCaMKII conformational change from CaM-dependence to CaM-independence. Furthermore, analyses using αCaMKII mutants showed that phosphorylation at T286 and T305/306 played positive and negative roles, respectively, during in vivo interaction with CaM and further suggested that CaM-dependent and CaM-independent conformational changed forms displays similar but distinct structures. CONCLUSIONS: Importantly, these structual differences between CaM-dependent and -independent forms of αCaMKII may exhibit differential functions for αCaMKII, such as interactions with other molecules required for LTP and memory. Our molecular probes could thus be used to identify therapeutic targets for cognitive disorders that are associated with the misregulation of αCaMKII.