Activation of the toll-like receptor 2 signaling pathway by GAPDH from bacterial strain RD055328.

We characterized the membrane vesicle fraction (RD-MV fraction) from bacterial strain RD055328, which is related to members of the genus Companilactobacillus and Lactiplantibacillus plantarum. RD-MVs and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were detected in the RD-MV fraction. Immunoglobulin A (IgA) was produced by Peyer's patch cells following the addition of the RD-MV fraction. In the presence of the RD-MV fraction, RAW264 cells produced the pro-inflammatory cytokine IL-6. Recombinant GAPDH probably induced the production of IL-6 by RAW264 cells via superficial toll-like receptor 2 (TLR2) recognition. A confocal laser scanning microscopy image analysis indicated that RD-MVs and GAPDH were taken up by RAW264 cells. GAPDH wrapped around RAW264 cells. We suggest that GAPDH from strain RD055328 enhanced the production of IgA by acquired immune cells via the production of IL-6 by innate immune cells through TLR2 signal transduction.

Enhancement of IgA production by membrane vesicles derived from Bifidobacterium longum subsp. infantis.

Immunoglobulin A (IgA) is involved in the maintenance of gut homeostasis. Although the oral administration of bifidobacteria increases the amount of fecal IgA, the effects of bifidobacteria on intestinal immunity remain unclear. We found and characterized membrane vesicles (MVs) derived from Bifidobacterium longum subsp. infantis toward host immune cells. Bifidobacterium infantis MVs consisted of a cytoplasmic membrane, and extracellular solute-binding protein (ESBP) was specifically detected. In the presence of B. infantis MVs or recombinant ESBP, RAW264 cells produced the pro-inflammatory cytokine IL-6. IgA was produced by Peyer's patches cells following the addition of B. infantis MVs. Therefore, ESBP of B. infantis MVs is involved in the production of IgA by acquired immune cells via the production of IL-6 by innate immune cells.

Influence of mutation in the regulatory domain of α-isopropylmalate synthase from Saccharomyces cerevisiae on its activity and feedback inhibition.

Isoamyl alcohol (i-AmOH) is produced from α-ketoisocaproate in the l-leucine biosynthetic pathway in yeast and controlled by the negative feedback regulation of α-isopropylmalate synthase (IPMS), which senses the accumulation of l-leucine. It is known that i-AmOH production increases when mutations in the regulatory domain reduce the susceptibility to feedback inhibition. However, the impact of mutations in this domain on the IPMS activity has not been examined. In this study, we obtained 5 IPMS mutants, encoding the LEU4 gene, N515D/S520P/S542F/A551D/A551V, that are tolerant to 5,5,5-trifluoro-dl-leucine. All mutant proteins were purified and examined for both IPMS activity and negative feedback activity by in vitro experiments. The results showed that not only the negative-feedback regulation by l-leucine was almost lost in all mutants, but also the IPMS activity was greatly decreased and the difference in IPMS activity among Leu4 mutants in the presence of l-leucine was significantly correlated with i-AmOH production.

The Novel Technique of Sternal Closure With Absorbable Mesh for Osteoporotic Patients.

After a median sternotomy, sternal instability can result in sternal infection. The usual sternal closure with stainless steel wires may result in sternal instability, especially in osteoporotic patients. An absorbable mesh (Super-FIXSORB-MX®40, Takiron Co Ltd, Osaka, Japan) for reinforcing the sternum has become commercially available. This paper reports a modified sternal closure procedure using this absorbable mesh in combination with heavy braided Polydiaxanon (PDS cord®, Ethicon, Inc., Somerville, NJ, USA) for osteoporotic patients.

Intramucosal colorectal carcinoma with lymphovascular invasion: clinicopathological characteristics of nine cases.

AIMS: Recent studies have provided the concept of invasive intramucosal colorectal carcinoma (CRC), and a case of intramucosal CRC with lymphatic invasion has been reported; however, the characteristics of such cases and the risk of lymph node metastasis have never been investigated. Therefore, we aimed to assess the pathological characteristics of intramucosal CRCs with lymphovascular invasion as well as the possibility of lymph node metastasis as an indication for additional surgery. METHODS AND RESULTS: To delineate the histological features of intramucosal CRCs with lymphovascular invasion, we analysed several histological features and compared their incidence among nine such cases, as well as 20 other cases of intramucosal CRCs without lymphovascular invasion. High-grade tumour budding and a pattern of 'eosinophilic cytoplasm and round nuclei with inflammatory reaction (ERI)' were morphological characteristics of intramucosal CRCs with lymphovascular invasion, compared with those without lymphovascular invasion (both P < 0.05). Among the seven lymph node-dissected cases of intramucosal CRCs with lymphovascular invasion, none showed lymph node metastasis. CONCLUSIONS: In intramucosal CRCs with lymphovascular invasion, high-grade tumour budding and the 'ERI' pattern are morphological characteristics that are distinct from those of non-invasive CRC, which is synonymous with high-grade dysplasia. Further studies using a larger number of cases by focusing on the above-mentioned histological pattern are expected to clarify the potential of lymph node metastasis of such cases.

Fascin as a Useful Marker for Identifying Neural Components in Immature Teratomas of Human Ovary and Those Derived From Murine Embryonic Stem Cells.

Immature teratoma of the human ovary is a rare disease, and its diagnosis and grading are currently based on histologic evaluation of the presence and amount of immature neural components in the tumor. Despite the importance of tumor grading, immature neural components especially without rosette formation are difficult to identify, partly because useful biomarkers for them are not yet available. Toward this goal, we investigated 16 immature teratomas from human ovaries as well as 10 of those derived from murine embryonic stem cells transplanted into immunodeficient mice. Immunohistochemistry was performed for cytokeratin, glial fibrillary acidic protein, S100, and fascin. It was demonstrated that glial fibrillary acidic protein and S100 expression was not observed in the immature neural components of immature teratomas derived from both human ovary and embryonic stem cells, although their expression was detected in mature neural tissues. In contrast, fascin immunopositivity was clearly found in both mature and immature neural components regardless of rosette formation in immature teratomas derived from both human ovary and embryonic stem cells. Assessment of immature neural components by fascin immunostaining yielded the same or slightly increased quantity than quantification based on hematoxylin and eosin staining. These results suggest that fascin immunostaining is useful as a biomarker in correctly diagnosing and grading human immature teratomas. Further, fascin immunostaining may contribute to the development of regenerative medicine through accurate assessment of the maturation status of pluripotent stem cell-derived tumors transplanted into immunodeficient mice.

Difference in immunohistochemical characteristics between Takayasu arteritis and giant cell arteritis: It may be better to distinguish them in the same age.

Objectives: This study aimed to compare Takayasu arteritis (TAK) with giant cell arteritis (GCA) through immunohistochemistry principally of inflammatory cells; these two disorders may be on the spectrum within a single disease state.Methods: Nine TAK and 5 GCA surgically resected vessel specimens were selected. TAK specimen was divided into each three acute-, chronic-, and healed-phase samples based on intimal and adventitial thickening. Immunohistochemical analysis was performed of smooth muscle cells and inflammatory cells including lymphocytes, plasma cells, macrophages, and dendritic cells, where three healed-phase TAK specimens were excluded due to paucity of inflammation. Immunopositive cells per three different fields in intima, media, and adventitia were counted in each specimen, and their numbers in these three layers along with total 3 layers were compared between the two disorders.Results: Intimal smooth muscle maturity estimated by ratio of h-Caldesmon+ cells to α-SMA+ cells significantly increased in chronic- and healed- over acute-phase increases in TAK. Mann-Whitney tests demonstrated significantly more adventitial lymphoplasmacytic infiltration and less intimal fascin+ dendritic cells, as well as overall more CD8+ T-cells, more CD20+ B-cells and lower CD4/8 ratio in TAK than in GCA.Conclusion: Different inflammatory involvement is suggested in the pathogenesis of TAK and GCA.

Vascular Smooth Muscle Cell Maturation Stage and Ki-67 Index are Diagnostic Biomarkers for Pathologic Grade of Ovarian Teratoma.

Although immature teratoma of the ovary is a rare disease, its pathologic grading, especially between low-grade (grade 1) and high-grade (grade 2 or 3) immature teratomas, is important for optimal therapy and prognosis. This grading, however, is currently solely dependent on quantitation of neuroepithelial components as judged by subjective assessments. As we have recently successfully studied the maturation of vascular smooth muscle cells (SMCs) in other organs using an h-caldesmon to α-smooth muscle actin (α-SMA) ratio, we decided to use this ratio to investigate a potential link between teratoma grade and SMC maturation, in combination with Ki-67 index. Sixteen immature teratomas along with 5 mature teratomas of the ovary were studied and stained with antibodies to CD31, α-SMA, h-caldesmon, and Ki-67. The number of vascular SMCs calculated using the α-SMA/CD31 ratio did not differ between teratoma grades (except between grade 0 and 3), whereas the number of mature vascular SMCs calculated using the h-caldesmon/CD31 ratio and maturation state calculated using the h-caldesmon/α-SMA ratio reduced significantly as teratoma grade progressed from 0 to 3. Furthermore, these parameters were significantly lower in high-grade than in low-grade immature teratomas (P<0.05). Ki-67 labeling index, regardless of germ cell layer, also significantly increased with teratoma grade (P<0.05). These results suggest that not only the number of neuroepithelial elements but also vascular immaturity and proliferating cell counts are biomarkers for ovarian teratoma grading. Thus, assessment of the maturity of vascular SMCs may serve as a valuable diagnostic tool for assessing teratoma maturity.

L-type amino acid transporter 1 expression in esophageal carcinogenesis according to WHO and Japanese classifications of intraepithelial neoplasia.

L-type amino acid transporter 1 (LAT1) has an essential role in cell proliferation especially in neoplasms. Although immunohistochemical expression of LAT1 has been investigated in invasive esophageal carcinoma, its expression in intraepithelial neoplasia (IEN) has not been reported. Further, classification of esophageal IEN is currently different between the World Health Organization (WHO) and Japanese criteria. Therefore, in this study, immunohistochemical expressions of LAT1 along with Ki-67 were analyzed in 66 esophageal samples of endoscopic submucosal dissection. Extension of cells positive for either marker within the epithelium, along with LAT1 intensity at the base of the epithelium, was evaluated. The results among early IENs, progressed IENs, and invasive carcinoma based on both WHO and Japanese criteria were compared. It was demonstrated that Ki-67+ cells extended toward the superficial layer in IENs, which was more pronounced in progressed compared with early IENs based on both WHO and Japanese criteria. Although similar results were obtained for LAT1+ cells, LAT1+ cell extended more in invasive carcinoma than in progressed IENs according to the WHO criteria. Further, LAT1 intensity was different between early and progressed IENs based on the Japanese criteria alone. Thus, use of LAT1 immunohistochemistry and Japanese classification may be more meaningful to characterize esophageal carcinogenesis.

Antifungal peptidic compound from the deep-sea bacterium Aneurinibacillus sp. YR247.

Aneurinibacillus: sp. YR247 was newly isolated from the deep-sea sediment inside the Calyptogena community at a depth of 1171 m in Sagami Bay. The strain exhibited antifungal activity against the filamentous fungus Aspergillus brasiliensis NBRC9455. A crude extract prepared from the YR247 cells by ethanol extraction exhibited broad antimicrobial activities. The antifungal compound is stable at 4-70 °C and pH 2.0-12.0. After treatment with proteinase K, the antifungal activity was not detected, indicating that the antifungal compound of strain YR247 is a peptidic compound. Electrospray ionization mass spectrometry of the purified antifungal compound indicated that the peptidic compound has an average molecular weight of 1167.9. The molecular weight of the antifungal compound from strain YR247 is different from those of antimicrobial peptides produced by the related Aneurinibacillus and Bacillus bacteria. The antifungal peptidic compound from the deep-sea bacterium Aneurinibacillus sp. YR247 may be useful as a biocontrol agent.

[Mitral Valve Repair in a Patient with Two Aneurysms of the Mitral Valve Due to Infective Endocarditis;Report of a Case].

Echocardiography revealed 2 aneurysms in the mitral valve with mitral regurgitation and aortic regurgitation,in a 42-years-old man. He had had infective endocarditis due to Streptococcus 9 months before. A prolapse in the right coronary cusp without vegetation and 2 aneurysms in the anterior mitral leaflet were found intraoperatively. Aortic valve was replaced, and then mitral aneurysms were resected and repaired using his own pericardium. There has been almost no regurgitation or recurrence of infection over 3 years.

Properties of an ionic liquid-tolerant Bacillus amyloliquefaciens CMW1 and its extracellular protease.

An ionic liquid-tolerant bacterium, Bacillus amyloliquefaciens CMW1, was isolated from a Japanese fermented soybean paste. Strain CMW1 grew in the presence of 10 % (v/v) 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), a commonly used ionic liquid. Additionally, strain CMW1 grew adequately in the presence of the hydrophilic ionic liquids 10 % (v/v) 1-ethyl-3-methylimidazolium trifluoromethanesulfonate ([EMIM]CF3SO3) or 2.5 % (v/v) 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([BMIM]CF3SO3). Strain CMW1 produced an extracellular protease (BapIL) in the culture medium. BapIL was stable in the presence of 80 % (v/v) ionic liquids, [EMIM]CF3SO3, [BMIM]Cl, [BMIM]CF3SO3, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate, and 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, and functioned in 10 % (v/v) these ionic liquids. BapIL was stable at pH 4.0-12.6 or in 4004 mM NaCl solution, and exhibited activity in the presence of 50 % (v/v) hydrophilic or hydrophobic organic solvents. BapIL was completely inhibited by 1 mM PMSF and partially by 5 mM EDTA. BapIL belongs to the true subtilisins according to analysis of the deduced amino acid sequence. We showed that BapIL from the ionic liquid-tolerant B. amyloliquefaciens CMW1 exhibited tolerance to ionic liquid and halo, alkaline, and organic solvents.

A high-molecular-weight, alkaline, and thermostable ß-1,4-xylanase of a subseafloor Microcella alkaliphila.

An endo ß-1,4-xylanase (XynE15) from a culture broth of a deep subseafloor microorganism, Microcella alkaliphila JAM-AC0309, was purified to homogeneity. The molecular mass of XynE15 was approximately 150 kDa as judged by SDS-PAGE. The optimal pH and temperature for hydrolysis of xylan were pH 8 and 65 °C. The enzyme was stable to incubation for 30 min at up to 75 °C, and the half-life at 50 °C was 48 h. XynE15 hydrolyzed arabinoxylan, oat spelt xylan, and birchwood xylan well, but not avicel, carboxymethylcellulose, or arabinan. Xylooligosaccharides were hydrolyzed to mainly xylobiose from higher than xylotetraose. The genome sequencing analysis of strain JAM-AC03039 revealed that XynE15 was composed of 1,319 amino acids with one catalytic domain and three carbohydrate-binding domains belonging to glycoside hydrolase (GH) family 10 and carbohydrate-binding module (CBM) family 4, respectively.

[Minimally Invasive Cardiac Surgery with Lower Partial Sternotomy for Combined Valve Disease;Report of a Case].

Combined valve surgery is usually performed with standard sternotomy although the efficacy and safety of single valve surgery with partial sternotomy has been established. We report a successful case of triple valve surgery with lower partial sternotomy. A 69-year-old woman underwent aortic valve replacement, mitral and tricuspid valve repair via lower partial sternotomy for moderate aortic and mitral valve regurgitation as well as severe tricuspid valve regurgitation. The operation was successfully performed with enough surgical field, without using any specific technical devises for minimally invasive cardiac surgery or blood transfusion. The ascending aorta, the superior vena cava and the right femoral vein were cannulated for cardiopulmonary bypass. The procedure was completed as in a conventional approach, except for a small incision for the femoral vein. This approach has several advantages;less trauma, less pain, earlier recovery, and better cosmetic outcomes. Triple valve surgery can be completed using lower partial sternotomy with benefits.

[Massive Pulmonary Hemorrhage During Cardiac Operation;Report of a Case].

The incidence of pulmonary hemorrhage during cardiac operation is not freqent but one of severe and lethal complications. We report a case of massive pulmonary hemorrhage, hemoptysis, suspected to be induced by a Swan-Ganz catheter. The patient had satisfactory progress postoperatively. An 80-year-old female patient underwent mitral valve replacement, tricuspid annuloplasty, and maze procedure. A Swan-Ganz catheter was inserted via the right jugular vein and fixed just at 40 cm as in usual preoperative induction. Operative procedures were uneventful, but active and massive airway hemorrhage started while weaning off cardiopulmonary bypass. A hematoma spreading under the visceral pleura of the right middle-lobe lung was found. We immediately replaced the endotracheal tube with a double-lumen one, and promptly decided to do lung lobectomy. These strategies were very helpful to rescue the patient, and led to her good recovery after the severe pulmonary hemorrhage, possibly induced by a catheter.

Young-age-onset pancreatoduodenal carcinoma in Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome, which is characterized by pancreatic fatty degeneration, skeletal growth retardation, and hematological dysfunction, is a congenital disease caused by SBDS gene mutations. Although hematological disorders often accompany this syndrome, carcinomas associated with this syndrome have not been reported except in one breast cancer and one moderately differentiated pancreatic cancer case. We report on an autopsy of a 24-year-old case of pancreatoduodenal carcinoma in Shwachman-Diamond syndrome. The histology of the tumor was undifferentiated carcinoma, which seems to have originated from either the pancreatic duct or the duodenal epithelium. The tumor was intermingled with two pathological changes characteristic of Shwachman-Diamond syndrome: fatty degeneration of the pancreas and inflammation of the villous stroma of the duodenum. Considering that SBDS protein regulates mitosis and its suppression causes genomic instability, this case might provide an example of carcinogenesis based on genomic instability, together with degenerative changes and chronic inflammation, at a very young age.

[Surgical technique of saphenous vein harvesting using a Cusco vaginal speculum].

We used Cusco vaginal speculum in harvesting saphenous vein graft (SVG) as an assist device for making a skin tunnel. After making 2 incisions of 3 to 4 cm, the SVG was dissected in a usual procedure. Then Cusco vaginal speculum was inserted into the skin tunnel between the 2 incisions. The SVG was dissected in a usual fashion under direct vision with the speculum. This procedure requires only small incisions, short learning curve and low cost. The new technique using Cusco vaginal speculum can be a reliable option for harvesting SVG.

Metabolic and Epigenetic Reprogramming in a Case of Nuclear Protein in Testis (NUT) Carcinoma of the Retroperitoneum.

Nuclear protein in testis (NUT) carcinoma is a rare but highly aggressive carcinoma, driven by genetic rearrangement of the NUT midline carcinoma family member 1 (NUTM1) gene on chromosome 15q14. Recently, a tight link has been suggested between genetic abnormalities and subsequent metabolic and epigenetic dysregulation to drive the progression of malignant tumors. However, it remains elusive whether such reprogramming could contribute to the pathogenesis of NUT carcinoma. We herein report an autopsy case of NUT carcinoma arising in the retroperitoneum of a 31-year-old male. Notably, reprogramming of glycolytic metabolism and epigenetic histone modifications was observed in this unusual NUT carcinoma case, and this phenomenon was further confirmed by an in vitro cell culture model with bromodomain containing 4 (BRD4)-NUT overexpression. The rationale for documenting the case is based on our findings to reveal that metabolic and epigenetic reprogramming could be one of the contributing factors to the pathogenesis of NUT carcinoma, which could be exploitable as a novel therapeutic target for this rare and aggressive cancer type.

DNA hypomethylator phenotype reprograms glutamatergic network in receptor tyrosine kinase gene-mutated glioblastoma.

DNA methylation is crucial for chromatin structure and gene expression and its aberrancies, including the global "hypomethylator phenotype", are associated with cancer. Here we show that an underlying mechanism for this phenotype in the large proportion of the highly lethal brain tumor glioblastoma (GBM) carrying receptor tyrosine kinase gene mutations, involves the mechanistic target of rapamycin complex 2 (mTORC2), that is critical for growth factor signaling. In this scenario, mTORC2 suppresses the expression of the de novo DNA methyltransferase (DNMT3A) thereby inducing genome-wide DNA hypomethylation. Mechanistically, mTORC2 facilitates a redistribution of EZH2 histone methyltransferase into the promoter region of DNMT3A, and epigenetically represses the expression of DNA methyltransferase. Integrated analyses in both orthotopic mouse models and clinical GBM samples indicate that the DNA hypomethylator phenotype consistently reprograms a glutamate metabolism network, eventually driving GBM cell invasion and survival. These results nominate mTORC2 as a novel regulator of DNA hypomethylation in cancer and an exploitable target against cancer-promoting epigenetics.