Label-Free Liquid Chromatography-Mass Spectrometry Quantitation of Relative N- and O-Glycan Concentrations in Human Milk in Japan.

Human milk is abundant in carbohydrates and includes human milk oligosaccharides (HMOs) and N/O-glycans conjugated to proteins. HMO compositions and concentrations vary in individuals according to the maternal secretor status based on the fucosyltransferase 2 genotype; however, the profile of N/O-glycans remains uninvestigated because of the analytical complexity. Herein, we applied a label-free chromatography-mass spectrometry (LC-MS) technique to elucidate the variation in the composition and concentration of N/O-glycans in human milk. We used label-free LC-MS to relatively quantify 16 N-glycans and 12 O-glycans in 200 samples of Japanese human milk (1-2 months postpartum) and applied high performance anion exchange chromatography with pulsed amperometric detection to absolutely quantify the concentrations of 11 representative HMOs. Cluster analysis of the quantitative data revealed that O-glycans and several HMOs were classified according to the presence or absence of fucose linked to galactose while N-glycans were classified into a different group from O-glycans and HMOs. O-glycans and HMOs with fucose linked to galactose were more abundant in human milk from secretor mothers than from nonsecretor mothers. Thus, secretor status influenced the composition and concentration of HMOs and O-glycans but not those of N-glycans in human milk.

Axillary lymph node pigmentation in tattooed patients.

Key Clinical Message: Tattooed pigments often migrate to the regional lymph nodes, leading to staining and swelling of these nodes. This migration can create confusion for surgeons when evaluating regional lymph nodes for staging of malignant disease like breast cancer. Abstract: A 50-year-old woman with left breast carcinoma underwent left mastectomy and sentinel lymph node (SLN) biopsy. A cherry blossom tattoo was observed on the left pectoral region, which she had acquired 6 years prior. Two lymph nodes were excised as SLNs and one of the two SLNs exhibited partial pink staining. Serial sections were performed on the block, revealing pinkish particles within the colored area on pathological examination. However, no metastasis was detected.

A soft fibroma of the nipple.

Although soft fibromas occur in the intertriginous area, including on the neck, axillae, and vulvovaginal locations, in rare cases, they can develop in the nipple. Doctors should consider soft fibroma as one of the differential diagnoses for nipple lesions.

An epidermal cyst in the areola.

Although epidermal cysts are common lesions of the scalp, face neck, and trunk, these cysts are rarely found in the areola. Doctors should think of epidermal cyst as one of differential diagnoses of an areolar lesions.

Large-scale preparation and glycan characterization of sialylglycopeptide from bovine milk glycomacropeptide and its bifidogenic properties.

Bovine glycomacropeptide (GMP) is a 7,000-Da glycopolypeptide released from κ-casein during cheese making. The O-glycan chains linked to GMP have many biological activities, but their utilization for nutraceutical products is limited due to their low content. To concentrate the functional glycan chains of GMP, we prepared sialylglycopeptide concentrate (SGC) from GMP-containing whey protein concentrate via proteolytic digestion of peptide chains and concentration of sialylglycopeptide by ultrafiltration using membranes with a molecular weight cut-off of 1,000 Da. The abundant saccharides detected in the prepared SGC were N-acetylneuraminic acid (Neu5Ac: 32.3% wt/wt), N-acetylgalactosamine (11.3%), and galactose (10.2%), which constitute O-glycans attached to GMP. The Neu5Ac content in SGC was found concentrated at approximately 4.8-fold of its content in GMP-containing whey protein concentrate (6.8%). Structural analysis of O-glycopeptides by liquid chromatography tandem mass spectrometry identified 88 O-glycopeptides. Moreover, O-acetylated or O-diacetylated Neu5Ac was detected in addition to the previously characterized O-glycans of GMP. Quantitative analysis of O-glycan in SGC by fluorescence labeling of chemically released O-glycan revealed that a disialylated tetrasaccharide was the most abundant glycan (76.6% of the total O-glycan). We further examined bifidogenic properties of SGC in vitro, which revealed that SGC served as a more potent carbon source than GMP and contributes to the growth-promoting effects on certain species of bifidobacteria. Overall, our study findings indicate that SGC contains abundant O-glycans and has a bifidogenic activity. Moreover, the protocol for the preparation of SGC described herein is relatively simple, providing a high yield of glycan, and can be used for large-scale preparation.

A case of long-term survival after splenectomy for solitary splenic metastasis from gastric cancer.

BACKGROUND: Very rarely does a splenic solitary metastasis arise from a gastric carcinoma because splenic metastasis is usually seen in association with widespread visceral metastasis. Splenectomy is considered to be a curative treatment; however, long-term prognosis after splenectomy has scarcely been reported. We report a case of a metachronous and solitary metastasis to the spleen from gastric cancer in which the patient achieved 5-year recurrence-free survival after splenectomy. CASE PRESENTATION: An 84-year-old man underwent an open total gastrectomy involving D1+ lymph nodes dissection for gastric cancer located in the cardia (pT3N1M0, pStage IIB). Eighteen months later, a 2-cm solitary hypodense lesion was detected in the spleen by computed tomography (CT). Twenty-three months later, the serum carcinoembryonic antigen (CEA) value elevated to 19.9 ng/ml, and abdominal CT revealed an increase in tumor size to 5 cm. Positron-emission tomography (PET)-CT revealed intense 18F-2-deoxy-2-fluoro-glucose (FDG) uptake in the spleen without the involvement of other organs and lymph nodes. We diagnosed him with solitary splenic metastasis from gastric cancer and performed a splenectomy 26 months after the first surgery. Histological examination revealed that the splenic tumor was a moderately differentiated adenocarcinoma, which was very similar to the primary gastric tumor; the lesion was diagnosed as a metastatic tumor from the previous gastric carcinoma. The patient remains healthy to date without recurrence, 5 years after the splenectomy. CONCLUSION: We experienced a case of a solitary splenic metastasis from gastric cancer in which 5-year recurrence-free survival was achieved after splenectomy. To determine the surgical indication in patients with splenic metastasis, it is important to differentiate between a solitary lesion or multiple metastasis. Especially, occult metastasis should be excluded by means of several months of follow-up with imaging tests and systemic FDG-PET surveys before splenectomy.

Alteration of Membrane Physicochemical Properties by Two Factors for Membrane Protein Integration.

After a nascent chain of a membrane protein emerges from the ribosomal tunnel, the protein is integrated into the cell membrane. This process is controlled by a series of proteinaceous molecular devices, such as signal recognition particles and Sec translocons. In addition to these proteins, we discovered two endogenous components regulating membrane protein integration in the inner membrane of Escherichia coli. The integration is blocked by diacylglycerol (DAG), whereas the blocking is relieved by a glycolipid named membrane protein integrase (MPIase). Here, we investigated the influence of these integration-blocking and integration-promoting factors on the physicochemical properties of membrane lipids via solid-state NMR and fluorescence measurements. These factors did not have destructive effects on membrane morphology because the membrane maintained its lamellar structure and did not fuse in the presence of DAG and/or MPIase at their effective concentrations. We next focused on membrane flexibility. DAG did not affect the mobility of the membrane surface, whereas the sugar chain in MPIase was highly mobile and enhanced the flexibility of membrane lipid headgroups. Comparison with a synthetic MPIase analog revealed the effects of the long sugar chain on membrane properties. The acyl chain order inside the membrane was increased by DAG, whereas the increase was cancelled by the addition of MPIase. MPIase also loosened the membrane lipid packing. Focusing on the transbilayer movement, MPIase reduced the rapid flip-flop motion of DAG. On the other hand, MPIase could not compensate for the diminished lateral diffusion by DAG. These results suggest that by manipulating the membrane lipids dynamics, DAG inhibits the protein from contacting the inner membrane, whereas the flexible long sugar chain of MPIase increases the opportunity for interaction between the membrane and the protein, leading to membrane integration of the newly formed protein.

CdsA is involved in biosynthesis of glycolipid MPIase essential for membrane protein integration in vivo.

MPIase is a glycolipid that is involved in membrane protein integration. Despite evaluation of its functions in vitro, the lack of information on MPIase biosynthesis hampered verification of its involvement in vivo. In this study, we found that depletion of CdsA, a CDP-diacylglycerol synthase, caused not only a defect in phospholipid biosynthesis but also MPIase depletion with accumulation of the precursors of both membrane protein M13 coat protein and secretory protein OmpA. Yeast Tam41p, a mitochondrial CDP-diacylglycerol synthase, suppressed the defect in phospholipid biosynthesis, but restored neither MPIase biosynthesis, precursor processing, nor cell growth, indicating that MPIase is essential for membrane protein integration and therefore for cell growth. Consistently, we observed a severe defect in protein integration into MPIase-depleted membrane vesicles in vitro. Thus, the function of MPIase as a factor involved in protein integration was proven in vivo as well as in vitro. Moreover, Cds1p, a eukaryotic CdsA homologue, showed a potential for MPIase biosynthesis. From these results, we speculate the presence of a eukaryotic MPIase homologue.

First report of the isolation of blaIMI-1-producing colistin-heteroresistant Enterobacter cloacae in Japan, September 2016.

The isolation of a carbapenem-resistant Enterobacter cloacae strain harboring the IMI-1 variant of blaIMI-1 carbapenemase points to the worldwide emergence of multidrug resistant bacteria as a potential source of health care infections. In this report, we describe the first isolation of E. cloacae with blaIMI-1 carbapenemase isolated from a Japanese patient in September 2016. The isolate was resistant to carbapenems, levofloxacin, and aminoglycosides, and heteroresistant to colistin but sensitive to fourth-generation cephalosporins. All microbiology laboratories worldwide should be made aware of these blaIMI-1-producing subtypes with unusual antibiotic susceptibility profiles.

Single nucleotide polymorphisms in genes encoding penicillin-binding proteins in ß-lactamase-negative ampicillin-resistant Haemophilus influenzae in Japan.

OBJECTIVE: ß-Lactamase-negative ampicillin-resistant Haemophilus influenzae is a common opportunistic pathogen of hospital- and community-acquired infections, harboring multiple single nucleotide polymorphisms in the ftsI gene, which codes for penicillin-binding protein-3. The objectives of this study were to perform comprehensive genetic analyses of whole regions of the penicillin-binding proteins in H. influenzae and to identify additional single nucleotide polymorphisms related to antibiotic resistance, especially to ampicillin and other cephalosporins. RESULTS: In this genome analysis of the ftsI gene in 27 strains of H. influenzae, 10 of 23 (43.5%) specimens of group III genotype ß-lactamase-negative ampicillin-resistant H. influenzae were paradoxically classified as ampicillin-sensitive phenotypes. Unfortunately, we could not identify any novel mutations that were significantly associated with ampicillin minimum inhibitory concentrations in other regions of the penicillin-binding proteins, and we reconfirmed that susceptibility to ß-lactam antibiotics was mainly defined by previously reported SNPs in the ftsI gene. We should also consider detailed changes in expression that lead to antibiotic resistance in the future because the acquisition of resistance to antimicrobials can be predicted by the expression levels of a small number of genes.

A novel detection procedure for mutations in the 23S rRNA gene of Mycoplasma pneumoniae with peptide nucleic acid-mediated loop-mediated isothermal amplification assay.

Rapid and easy detection of a single nucleotide point mutation of bacterial genes, which is directly linked to drug susceptibility, is essential for the proper use of antimicrobial agents. Here, we established a detection method using a peptide nucleic acid mediated loop-mediated amplification (LAMP) assay for macrolide (ML)-susceptible Mycoplasma pneumoniae. This assay specifically detected the absence of missense mutations encoding the central loop of domain V in the gene encoding 23S rRNA, which can reduce the affinity for MLs and subsequently generate ML-resistant strains of M. pneumoniae. Reactions were performed at 62°C for 60min and targeted gene amplifications were detected by real-time turbidity with a turbidimeter and naked-eye inspection of a color change. The assay had an equivalent detection limit of 100.0fg of DNA with the turbidimeter and showed specificity against 54 types of pathogens, whereas amplification was completely blocked, even at 100.0pg of DNA per reaction, in the presence of point mutations at 2063A and 2064A. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. This method would be a simple and rapid protocol for single nucleotide polymorphism genotyping as point-of-care testing technology without amplification of the sequences carrying the point mutations 2063A and 2064A in ML-resistant M. pneumoniae strains.

Orientation and Order of the Amide Group of Sphingomyelin in Bilayers Determined by Solid-State NMR.

Sphingomyelin (SM) and cholesterol (Chol) are considered essential for the formation of lipid rafts; however, the types of molecular interactions involved in this process, such as intermolecular hydrogen bonding, are not well understood. Since, unlike other phospholipids, SM is characterized by the presence of an amide group, it is essential to determine the orientation of the amide and its order in the lipid bilayers to understand the nature of the hydrogen bonds in lipid rafts. For this study, 1'-(13)C-2-(15)N-labeled and 2'-(13)C-2-(15)N-labeled SMs were prepared, and the rotational-axis direction and order parameters of the SM amide in bilayers were determined based on (13)C and (15)N chemical-shift anisotropies and intramolecular (13)C-(15)N dipole coupling constants. Results revealed that the amide orientation was minimally affected by Chol, whereas the order was enhanced significantly in its presence. Thus, Chol likely promotes the formation of an intermolecular hydrogen-bond network involving the SM amide without significantly changing its orientation, providing a higher order to the SM amide. To our knowledge, this study offers new insight into the significance of the SM amide orientation with regard to molecular recognition in lipid rafts, and therefore provides a deeper understanding of the mechanism of their formation.

Centerband-only analysis of rotor-unsynchronized spin echo for measurement of lipid (31) P chemical shift anisotropy.

Structural diversity and molecular flexibility of phospholipids are essential for biological membranes to play key roles in numerous cellular processes. Uncovering the behavior of individual lipids in membrane dynamics is crucial for understanding the molecular mechanisms underlying biological functions of cell membranes. In this paper, we introduce a simple method to investigate dynamics of lipid molecules in multi-component systems by measuring the (31) P chemical shift anisotropy (CSA) under magic angle spinning (MAS) conditions. For achieving both signal separation and CSA determination, we utilized a centerband-only analysis of rotor-unsynchronized spin echo (COARSE). This analysis is based on the curve fitting of periodic modulation of centerband intensity along the interpulse delay time in rotor-unsynchronized spin-echo experiments. The utility of COARSE was examined by using phospholipid vesicles, a three-component lipid raft model system, and archaeal purple membranes. We found that the apparent advantages of this method are high resolution and high sensitivity given by the moderate MAS speed and the one-dimensional acquisition with short spin-echo delays. COARSE provides an alternative method for CSA measurement that is effective in the investigation of lipid polymorphologies.

[A multicenter study of the antimicrobial susceptibility of Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae, and Moraxella catarrhalis from community acquired infections in Saitama, Japan].

We examined regional surveillance of antimicrobial susceptibility of community acquired bacterial pathogens from patients in Saitama, Japan. The fourth-year survey was conducted in three of the period 2007-2010 (period I, 2007-2008; period II, 2008-2009; period III, 2009-2010). Antimicrobial susceptibility testing was conducted at the central reference laboratory according to the method recommended by Japanese Society of Chemotherapy using maximum 13 antibacterial agents. Susceptibility testing was evaluable with 789 strains (227 Streptococcus pneumoniae, 148 Streptococcus pyogenes, 220 Haemophilus influenzae, and 194 Moraxella catarrhalis). Ratio of penicillin-susceptible S. pneumoniae (PSSP, MIC of benzylpenicillin ≤ 0.06 µg/mL) was 43.5% (period I), 43.5% (period II) and 55.8% (period III), and those of erythromycin-sensitive and azithromycin-sensitive S. pyogenes were 100% and 65.5% (period I), 47.9% and 47.9% (period II), 29.4%, and 29.4% (period III) , respectively. Among H. influenzae, ß-lactamase-nonproducing ampicillin-resistant isolates were 34.9% (period I), 25.8% (period II), and 17.1% (period III); however, ß-lactamase-nonproducing ampicillin-intermediately resistant isolates were 19.8% (period I), 26.9% (period II), and 29.3% (period III). Regarding M. catarrhalis, macrolides showed potent activities, with MIC90s of ≤ 0.25-0.5 µg/mL, and fluoroquinolones showed strong activities, with MIC90s ≤0.03-0.125 µg/mL. The result of this survey indicated that the trends observed were similar to the results of previous nationwide surveillance.

[Trend of susceptibility testing of clinical isolates of Candida species from aseptic samples in hospitals in Saitama prefecture].

We investigated the susceptibility of Candida species from clinical aseptic samples, including blood, at some hospitals in Saitama prefecture. Candida spp. detected from aseptic samples in the 6 institutes in Saitama prefecture from November 2007 to July 2011 were studied. The number of isolates was 85, which are 43 (50.6%) of Candida albicans, 24 (28.2%) of Candida parapsilosis, 5 (5.9%) of Candida glabrata, 5 (5.9%) of Candida tropicalis, 4 (4.7%) of Candida guilliermondii, 2 (2.4%) of Candida fermentati, 1 (1.2%) of Candida famata and Candida lusitaniae, respectively. All isolates were susceptible to amphotericin B. However, resistant isolates against micafungin were 3 in 5 of C. glabrata. We analyzed susceptibility of Candida spp. in Saitama prefecture in the article, and our study might be useful for the fungal therapy in the region.

Epidermal cyst of the breast treated by vacuum-assisted biopsy.

A 38-year-old woman presented for evaluation of a firm mass (measuring 20 × 20 mm) in the upper outer quadrant of her left breast. On the basis of the clinical and radiologic findings, we diagnosed a benign breast tumor and scheduled removal by a hand-held vacuum-assisted biopsy device (VABD) under ultrasonographic guidance. Because the first specimen removed from the tumor was white, flaky, and waxy material, we strongly suspected that the lesion was an epidermal cyst. We continued VABD treatment until the tumor was invisible on real-time ultrasonography. Histologic examination demonstrated that the tumor was composed of mature stratified squamous epithelium and laminated layers of keratin, findings consistent with a diagnosis of epidermal cyst. These cysts rarely occur in the breast and are sometimes difficult to distinguish from breast cancer. To our knowledge, this is the first report of an epidermal cyst treated by VABD.

A first report of Rothia aeria endocarditis complicated by cerebral hemorrhage.

We herein report the first case of infective endocarditis attributable to Rothia aeria, which had a fatal outcome after cerebral hemorrhagic infarction and was not susceptible to vancomycin. If Gram-positive bacillary or filamentous bacteria that form white, coarse, dry colonies are detected, keeping the possibility of Rothia species in mind is advisable because members of this species can cause severe infections.