External Ear Melanoma Treated with Auricular Reconstruction Using Four Different Tissues in a 16-Year-Old Patient.

The auricular region is a common site of cutaneous malignancies, most of which are nonmelanoma skin cancers, such as basal cell carcinoma and squamous cell carcinoma, in older patients. They are often treated with limited surgery that can be performed under local anesthesia. We report the case of a young patient with external ear melanoma who underwent reconstruction for defects of more than one-half of the helix and concha using four types of tissues: a rib cartilage graft, temporoparietal fascia flap, full-thickness skin graft, and retroauricular flap. Extending the retroauricular flap posteriorly to the entire hairless area allowed us to cover the anterior surface of the rib cartilage framework, which helped effectively achieve aesthetics. In auricle reconstruction, it is crucial to determine how well the anterior surface of the auricle is created.

Novel multicellular prokaryote discovered next to an underground stream.

A diversity of prokaryotes currently exhibit multicellularity with different generation mechanisms in a variety of contexts of ecology on Earth. In the present study, we report a new type of multicellular bacterium, HS-3, isolated from an underground stream. HS-3 self-organizes its filamentous cells into a layer-structured colony with the properties of a nematic liquid crystal. After maturation, the colony starts to form a semi-closed sphere accommodating clusters of coccobacillus daughter cells and selectively releases them upon contact with water. This is the first report that shows that a liquid-crystal status of cells can support the prokaryotic multicellular behavior. Importantly, the observed behavior of HS-3 suggests that the recurrent intermittent exposure of colonies to water flow in the cave might have been the ecological context that cultivated the evolutionary transition from unicellular to multicellular life. This is the new extant model that underpins theories regarding a role of ecological context in the emergence of multicellularity.

Metachronous Liver Metastasis from Alpha-Fetoprotein-Producing Gastric Cancer Successfully Treated with Capecitabine/Oxaliplatin Combination Chemotherapy.

A consensus regarding the treatment of recurrent alpha-fetoprotein-producing gastric carcinoma due to its rarity is lacking. We herein describe a case of such a carcinoma that was associated with metachronous liver metastasis. A 73-year-old man was referred for the surgical treatment of a type 2 gastric tumor that extended from the greater curvature of the gastric corpus to the pylorus. As no remote metastases were detected, the patient underwent open total gastrectomy with lymphadenectomy and Roux-en-Y reconstruction. Histopathological examination revealed regional lymph node metastasis and the invasion of the muscularis propria by a moderately differentiated adenocarcinoma. Immunostaining of the primary tumor was positive for alpha-fetoprotein and negative for human epidermal growth factor receptor 2. Serum alpha-fetoprotein levels decreased to within normal limits after eight courses of S-1 monotherapy; however, levels started to increase, and a hypovascular nodule in segment 5/6 of the liver was detected 3.5 years later. Serum alpha-fetoprotein levels returned to the normal range, and the tumor was undetectable after four courses of capecitabine and oxaliplatin therapy. No recurrence was detected at 1.5-year follow-up. This case demonstrates that combined capecitabine and oxaliplatin therapy can successfully treat metachronous liver metastasis from alpha-fetoprotein-producing gastric carcinoma.

Lower Eyelid Reconstruction Using a Myotarsocutaneous Flap while Considering the Superior and Inferior Palpebral Sulci.

Various reconstructive procedures have been reported for full-thickness defects of the lower eyelid after tumor excision or trauma. In eyelid reconstruction, not only functionality but also cosmetic results are important; furthermore, making scars inconspicuous is challenging. The purpose of this study is to make the scars less noticeable in lower eyelid reconstruction. We reconstructed the anterior lamella using a myotarsocutaneous flap and the posterior lamella of the donor site using a palatal mucosal graft in a 61-year-old man with basal cell carcinoma of the lower eyelid. In designing the myotarsocutaneous flap, we matched the upper edge of the flap with the superior palpebral sulcus, and the lower edge with the inferior palpebral sulcus. The flap length was the same as the width of the defect. The lateral side of the postoperative scar was hidden by the excess skin of the upper eyelid, while the caudal side of the scar and the trapdoor deformity was covered with a tear trough. After movement, the flap was not located outside the lateral canthus, and good cosmetic results were obtained. This report describes our surgical procedure.

Curry Leaf Triggers Cell Death of P. gingivalis with Membrane Blebbing.

Periodontal disease has become a serious public health problem, as indicated by accumulating evidence that periodontal disease is not only a major cause of tooth loss but is also associated with various systemic diseases. The present study assessed the anti-bacterial activities of three herbal products (curry leaf, clove, and cinnamon) against Porphyomonas gingivalis, a keystone pathogen for periodontal diseases. The curry leaf extract (CLE) showed the strongest growth inhibitory activity among them, and the activity was maintained even after extensive heat treatment. Of note, while clove and cinnamon extracts at sub-minimum inhibitory concentrations (sub-MICs) significantly enhanced the biofilm formation of P. gingivalis, CLE at sub-MIC did not have any effect on the biofilm formation. The MIC of CLE against P. gingivalis was higher than those against a wide range of other oral bacterial species. P. gingivalis cells were completely killed within 30 min after treatment with CLE. Spatiotemporal analysis using high-speed atomic force microscopy revealed that CLE immediately triggered aberrant membrane vesicle formation on the bacterial surface. Bacterial membrane potential assay revealed that CLE induced depolarization of the bacterial membrane. Taken together, these findings suggest the mechanism behind early bactericidal activity of CLE and its therapeutic applicability in patients with periodontal diseases.

Two-way Revascularization to Manage Celiac Artery Stenosis during Pancreaticoduodenectomy: A Case Report.

Celiac artery (CA) occlusion, or stenosis, is not uncommon, and most cases are asymptomatic. If the CA is occluded, collateral circulation from the superior mesenteric artery (SMA) is maintained through the pancreaticoduodenal arcade. However, the pancreaticoduodenal arcade is removed if pancreaticoduodenectomy (PD) is performed, which results in ischemia of the liver, stomach, and residual pancreas. Thus, these patients require CA revascularization, which can include antegrade endovascular reconstruction and retrograde reconstruction using vascular anastomosis from the SMA system to the CA system. Both methods carry risks of restenosis or anastomotic thrombosis. We report a technique that involves a combination of both revascularization methods in an 89-year-old man who underwent PD for lower bile duct cancer. Preoperative endovascular stent placement in the CA preserved antegrade blood flow to the liver, and intraoperative vascular anastomosis of the jejunal artery and right gastroepiploic artery achieved retrograde blood flow. Although we confirmed both stent and anastomosis patency and blood circulation in our case, obstruction of 1 of these revascularization pathways would not likely lead to ischemia of the liver. Thus, our 2-way revascularization technique for managing celiac artery stenosis during PD may reduce the risk of organ ischemia.

Steric microstructure of mixed-species biofilm formed by interaction between Lactobacillus plantarum ML11-11 and Saccharomyces cerevisiae.

The mixed-species biofilm of Lactobacillus plantarum ML11-11 (LAB) and yeast had a double-layered structure with the ground layer composed of LAB cells, and the upper layer composed of coaggregates of LAB and yeast cells. The ability of LAB to adhere to both, the solid surface and the yeast cells, enabled the formation and maintenance of the biofilm as an ecosystem for LAB and yeast.

Microbial behavior and changes in food constituents during fermentation of Japanese sourdoughs with different rye and wheat starting materials.

Sourdough is a food item made by kneading grain flour and water together and allowing fermentation through the action of lactic acid bacteria (Lactobacillales) and yeast. Typically, Japanese bakeries make sourdough with rye flour, wheat flour, malt extract, and water and allow spontaneous fermentation for 6 days. We compared the microbial behavior and food components, such as organic acids, sugars, and free amino acids, of sourdoughs made using two different rye and wheat flours during the 6-day fermentation period. Comparisons were made for two types of rye and wheat flours, using different production sites and different milling, distribution, and storage conditions. The microbial count was evaluated using different culture media. All sourdough types showed a significant increase in lactic acid levels on fermentation day 2 and a decrease in free amino acid levels on day 4. Low overall lactic acid production and little fluctuation in sugar levels occurred in sourdough made from French ingredients. For sourdough made from Japanese ingredients, sugar levels (chiefly glucose, sucrose, and maltose) declined on fermentation day 1, increased on day 2, and declined by day 5. With the French ingredients, no yeast cells were detected until day 3, and many acid precursors of sourdough flavor components were detected. Yet with the Japanese ingredients, 106/g yeast cells were detected on days 3-5, as well as sourdough-flavor esters and alcohols. Differences in raw material quality affected the microbial behavior and changes in food constituents during the fermentation process and, consequently, the sourdough flavor.

Increase in hydrophobicity of Bacillus subtilis spores by heat, hydrostatic pressure, and pressurized carbon dioxide treatments.

The effects of heat treatment (HT), hydrostatic pressure treatment (HPT), and pressurized carbon dioxide treatment (CT) on surface hydrophobicity of B. subtilis 168 spores were investigated. The spore surface hydrophobicity was measured by determining the ratio of hydrophobic spores (RHS) that were partitioned into the n-hexadecane phase from the aqueous spore suspension. The RHS after HT generally increased in a temperature-dependent manner and reached approximately 10% at temperatures above 60°C. The effects of pressurization by HPT and accompanying temperature on increased RHS were complex. The highest RHS after HPT was approximately 17%. Following CT, RHS reached approximately 80% at 5 MPa at 80°C for 30 min. An increased treatment temperature enhanced RHS by CT. The increase in RHS by CT led to the formation of spore clumps and adhesion of spores to hydrophobic surfaces. Acidification of spore suspension to pH 3.2, expected pH during CT, by HCl also increased the adhesion of spores at the similar degree with CT. The spore surface zeta potential distribution was not changed by CT. Furthermore, spores with increased RHS after CT had germination-like phenomena including loss of their refractility and enhanced staining by 4',6-diamidino-2-phenylindole. Physiological germination that was induced by the addition of l-alanine also increased the RHS. From these results, it is clear that CT under heating considerably increases RHS. CT under heating considerably increases RHS. This increase in RHS may be due to acidification or germination-like phenomena during CT.

Analysis of transcription factors expressed at the anterior mouse limb bud.

Limb bud patterning, outgrowth, and differentiation are precisely regulated in a spatio-temporal manner through integrated networks of transcription factors, signaling molecules, and downstream genes. However, the exact mechanisms that orchestrate morphogenesis of the limb remain to be elucidated. Previously, we have established EMBRYS, a whole-mount in situ hybridization database of transcription factors. Based on the findings from EMBRYS, we focused our expression pattern analysis on a selection of transcription factor genes that exhibit spatially localized and temporally dynamic expression patterns with respect to the anterior-posterior axis in the E9.5-E11.5 limb bud. Among these genes, Irx3 showed a posteriorly expanded expression domain in Shh-/- limb buds and an anteriorly reduced expression domain in Gli3-/- limb buds, suggesting their importance in anterior-posterior patterning. To assess the stepwise EMBRYS-based screening system for anterior regulators, we generated Irx3 transgenic mice in which Irx3 was expressed in the entire limb mesenchyme under the Prrx1 regulatory element. The Irx3 gain-of-function model displayed complex phenotypes in the autopods, including digit loss, radial flexion, and fusion of the metacarpal bones, suggesting that Irx3 may contribute to the regulation of limb patterning, especially in the autopods. Our results demonstrate that gene expression analysis based on EMBRYS could contribute to the identification of genes that play a role in patterning of the limb mesenchyme.

Studies on formation, control and application of biofilm formed by food related microorganisms.

Biofilms are sessile microbial aggregates on the interfaces, and they were usually considered as microbial contamination sources in medical care and various industries. We studied the control and application of biofilms formed by food-related microorganisms, and mechanism of the biofilm formation was also investigated. We studied the biofilm formation in mixed cultures using various combinations of two strains of food-related microorganisms. There were various microorganisms that showed decreased or increased biofilm formation in the mixed culture in comparison with that in a single culture. Biofilm formed by lactic acid bacteria and yeast isolated from traditional fermented food, Fukuyama pot vinegar, exhibited unique feature in that structure and formation mechanism, and expected to be used as an immobilized microorganism in fermentation production. Here our studies on the control and application of biofilms and the mechanisms of its formation were described.

Awa1p on the cell surface of sake yeast inhibits biofilm formation and the co-aggregation between sake yeasts and Lactobacillus plantarum ML11-11.

We examined mixed-species biofilm formation between Lactobacillus plantarum ML11-11 and both foaming and non-foaming mutant strains of Saccharomyces cerevisiae sake yeasts. Wild-type strains showed significantly lower levels of biofilm formation compared with the non-foaming mutants. Awa1p, a protein involved in foam formation during sake brewing, is a glycosylphosphatidylinositol (GPI)-anchored protein and is associated with the cell wall of sake yeasts. The AWA1 gene of the non-foaming mutant strain Kyokai no. 701 (K701) has lost the C-terminal sequence that includes the GPI anchor signal. Mixed-species biofilm formation and co-aggregation of wild-type strain Kyokai no. 7 (K7) were significantly lower than K701 UT-1 (K701 ura3/ura3 trp1/trp1), while the levels of strain K701 UT-1 carrying the AWA1 on a plasmid were comparable to those of K7. The levels of biofilm formation and co-aggregation of the strain K701 UT-1 harboring AWA1 with a deleted GPI anchor signal were similar to those of K701 UT-1. These results clearly demonstrate that Awa1p present on the surface of sake yeast strain K7 inhibits adhesion between yeast cells and L. plantarum ML11-11, consequently impeding mixed-species biofilm formation.

Acid resistance contributes to the high-pressure carbon dioxide resistance of Escherichia coli K-12.

Effect of deletion of acid resistant genes of E. coli on the high-pressure carbon dioxide (HPC) resistance was investigated. Genes coding amino acid decarboxylases, such as lysine, arginine, and glutamate decarboxylase, were found to contribute to HPC resistance. Protonophore-treated cells showed hypersensitivity to HPC, confirming that HPC induced cytoplasm acidification and exerted severe damage on cells by intrusion of gaseous carbon dioxide into cytoplasm.

Screening of lactic acid bacteria that can form mixed-species biofilm with Saccharomyces cerevisiae.

The abilities of lactic acid bacteria (LAB) to form mixed-species biofilm with Saccharomyces cerevisiae in a static co-culture were investigated out of 168 LAB stock cultures, and two Lactobacillus plantarum strains (D71 and E31) and one Leuconostoc mesenteroides strain K01 were found to form mixed-species biofilm with S. cerevisiae BY4741. SEM observation showed that there was no significant difference in morphological properties among these three mixed-species biofilms and they resembled that formed by S. cerevisiae with L. plantarum ML11-11 previously isolated from a brewing sample of Fukuyama pot vinegar. The co-aggregation assays showed that L. plantarum D71 and L. plantarum E31 could co-aggregate with S. cerevisiae similarly to L. plantarum ML11-11, while L. mesenteroides K01 had no ability to co-aggregate with yeast. The above results indicate that aggregation followed by direct cell-to-cell contact is required for mixed-species biofilm formation between these L. plantarum strains and S. cerevisiae, though some different mechanism may be involved in biofilm formation between L. mesenteroides strain and S. cerevisiae.

Fimbriae and lipopolysaccharides are necessary for co-aggregation between Lactobacilli and Escherichia coli.

Cells of Lactobacilli co-aggregated with Escherichia coli K-12 cells to form co-aggregates under mixed-culture conditions at 37 °C for 24 h. Co-aggregation was inhibited by sodium dodecyl sulfate but not by protease. E. coli deletion mutants of fimbriae formation and lipopolysaccharide (LPS) formation did not co-aggregate with Lactobacilli. These results showed that fimbriae and LPS are necessary for co-aggregation between Lactobacilli and E. coli.

Manganese Ion Increases LAB-yeast Mixed-species Biofilm Formation.

Remarkable LAB-yeast mixed-species biofilm was formed by lactic acid bacteria (LAB) Lactobacillus plantarum ML11-11 isolated from Fukuyama pot vinegar and Saccharomyces cerevisiae. This mixed-species biofilm formation increased in proportion to the YPD medium concentration but decreased in proportion to the MRS medium concentration. The effect of MRS components on mixed-species biofilm formation was investigated in a YPD medium environment, and it was clarified that beef extract (one of the MRS medium components) decreased mixed-species biofilm formation. On the other hand, manganese sulfate (another component in MRS) remarkably increased both LAB single- and LAB-yeast mixed-species biofilm formation. LAB single- and mixed-species biofilm formation were increased in proportion to the manganese sulfate concentration up to 1 mM and 100 µM, respectively. The growth of L. plantarum ML11-11 was increased significantly by the addition of 10 µM manganese sulfate and was resistant to higher concentration of up to 100 mM, but growth of S. cerevisiae was sensitive to manganese ion above 100 µM. These results suggested that mixed-species biofilm formation could be controlled artificially by controlling the manganese ion level.

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.

Antimicrobial activity of the carnivorous plant Dionaea muscipula against food-related pathogenic and putrefactive bacteria.

Solvent extracts from the carnivorous plant Dionaea muscipula (Venus flytrap) were prepared using eight different organic solvents, and examined for antibacterial activity against food-related pathogenic and putrefactive bacteria. All solvent extracts showed higher antibacterial activity against gram positive bacteria than against gram negative bacteria. The TLC-bioautography analysis of the extracts revealed that a yellow spot was detected at Rf value of 0.85, which showed strong antibacterial activity. The UV, MS, and NMR analyses revealed that the antibacterial compound was plumbagin.

Yeasts and lactic acid bacteria mixed-specie biofilm formation is a promising cell immobilization technology for ethanol fermentation.

We previously found that some Saccharomyces cerevisiae and Lactobacillus plantarum remarkably formed mixed-specie biofilm in a static co-culture and deduced that this biofilm had potential as immobilized cells. We investigated the application of mixed-specie biofilm formed by S. cerevisiae BY4741 and L. plantarum HM23 for ethanol fermentation in repeated batch cultures. This mixed-specie biofilm was far abundantly formed and far resistant to washing compared with S. cerevisiae single biofilm. Adopting mixed-specie biofilm formed on cellulose beads as immobilized cells, we could produce enough ethanol from 10 or 20 % glucose during ten times repeated batch cultures for a duration of 10 days. Cell numbers of S. cerevisiae and L. plantarum during this period were stable. In mixed-specie biofilm system, though ethanol production was slightly lower compared to S. cerevisiae single-culture system due to by-production of lactate, pH was stably maintained under pH 4 without artificial control suggesting high resistance to contamination. Inoculated model contaminants, Escherichia coli and Bacillus subtilis, were excluded from the system in a short time. From the above results, it was indicated that the mixed-specie biofilm of S. cerevisiae and L. plantarum was a promising immobilized cell for ethanol fermentation for its ethanol productivity and robustness due to high resistance to contamination.

Significance of microbial symbiotic coexistence in traditional fermentation.

Symbiosis has long been a central theme in microbiology. There have been many studies on the symbioses between microorganisms and higher organisms such as plants and animals. There also have been some studies on the symbiosis or coexistence of microorganisms, such as yeasts, lactic acid bacteria (LAB), acetic acid bacteria (AAB) and koji molds, in traditional fermentation (brewing). These microorganisms are considered to interact and cooperate with each other in various natural environments, such as dropped cereal crops or ripe fruits. Human beings have taken advantage of these microbial interactions for producing various fermented foods.