Effect of phosphoproteins on intracellular calcification of bacteria.

This study aimed to evaluate the effects of phosphoproteins on bacterial mineralization. Dental calculus formation is attributed to bacterial mineralization in the oral cavity; however, the influence of phosphoproteins (which are abundant in saliva) is not clear. The model bacterium Escherichia coli was suspended in a calcification solution containing casein as a model phosphoprotein. To evaluate mineralization independent of bacterial metabolism, bacteria killed by heat treatment at 70°C were compared with viable bacteria. After incubation at 37°C for 24 h, the mode of calcification was observed using electron microscopy and energy dispersive x-ray spectroscopy. Solutions without casein produced precipitation in solution, which was identical to that in experiments without bacteria. In contrast, calcification solutions with 200 ppm casein only produced calcium phosphate deposition intracellularly. Without heat treatment, intracellular calcification rarely occurred, even when casein was added. Thus, phosphoproteins promoted intracellular calcification of dead bacteria; this is similar to the calcification of insoluble matrices, such as collagen fibrils, promoted by acidic polymers. We concluded that intracellular calcification is caused by the collagen fibril-like behavior of dead bacteria. The promotion of intracellular calcification of dead bacteria by phosphoproteins suggested a basic principle of dental calculus formation.

Bacteriologically Determined De Novo Tuberculosis during Tumor Necrosis Factor-α Inhibitor Therapy.

A 58-year-old man with Crohn's disease received adalimumab for 13 months after screening results for tuberculosis were found to be negative. He was diagnosed with de novo mediastinal lymph-node tuberculosis, which was proved to be bacteriologically identical to that of an individual with smear positive lung tuberculosis by a variable number of tandem repeat analyses. After initiating anti-tuberculosis therapy, the patient developed immune reconstitution syndrome, which was improved by the re-administration of adalimumab. Even in countries with an intermediate tuberculosis burden, including Japan, we need to be alert for de novo tuberculosis as well as its reactivation during tumor necrosis factor-α inhibitor therapy.

Insights into the ubiquinol/dioxygen binding and proton relay pathways of the alternative oxidase.

The alternative oxidase (AOX) is a monotopic diiron carboxylate protein which catalyzes the four-electron reduction of dioxygen to water by ubiquinol. Although we have recently determined the crystal structure of Trypanosoma brucei AOX (TAO) in the presence and absence of ascofuranone (AF) derivatives (which are potent mixed type inhibitors) the mechanism by which ubiquinol and dioxygen binds to TAO remain inconclusive. In this article, ferulenol was identified as the first competitive inhibitor of AOX which has been used to probe the binding of ubiquinol. Surface plasmon resonance reveals that AF is a quasi-irreversible inhibitor of TAO whilst ferulenol binding is completely reversible. The structure of the TAO-ferulenol complex, determined at 2.7 Å, provided insights into ubiquinol binding and has also identified a potential dioxygen molecule bound in a side-on conformation to the diiron center for the first time.

Evaluation of Quality of Life in Japanese Glaucoma Patients and its Relationship With Visual Function.

PURPOSE: We assessed vision-related quality of life (VR-QOL) in glaucoma patients and examined the relationship between VR-QOL and indices of visual function and subjective symptoms. PATIENTS AND METHODS: Four hundred seventy-two glaucoma patients completed a questionnaire concerning VR-QOL and subjective symptoms of glaucoma. VR-QOL was based on 5 subscales selected from the National Eye Institute 25-item Visual Function Questionnaire (NEI VFQ-25), whereas subjective symptoms were examined using original questions that we prepared. The following were selected as visual function indices: corrected visual acuity measured with a distance visual acuity chart, mean deviation scores measured with a static perimeter, and 4 threshold levels in the innermost center of a visual field (4 pericentral test points). Disease staging was conducted according to the Aulhorn classification system. Correlations of visual function with VR-QOL were evaluated using the Spearman test. RESULTS: Correlations were observed in almost all subscales of the NEI VFQ-25, including correlations with corrected vision, disease stage, mean deviation score, and minimum threshold values of the 4-point pericentral test on the temporal side (P<0.0001). Visual function indices affecting driving differed between daytime and night driving (P<0.0001). NEI VFQ-25 subscales were correlated with subjective symptoms felt by the patients while driving (P<0.0001). CONCLUSIONS: Minimum threshold values of the 4 pericentral test points on the temporal side are useful for the assessment of VR-QOL. Minimum threshold values of the 4 pericentral test points on the temporal side are useful for assessing visual field abnormalities that are associated with decreased VR-QOL.

[A Case of Radical Resection of Rectal Cancer with Multiple Liver and Lung Metastases after Preoperative Chemotherapy].

We report a case of radical resection of rectal cancer with multiple liver and lung metastases after preoperative chemotherapy. A 54-year-old woman presented with abdominal pain and loss of body weight due to rectal cancer with multiple liver and lung metastases. Therefore, the patient received 14 courses of bevacizumab+mFOLFOX6, and 7 courses of panitumumab+FOLFIRI. After the chemotherapy, the size of the distant metastases reduced by 62% on computed tomography, according to RECIST. Due to the reduction in size, a conversion surgery was attempted. First, an abdominal operation with laparoscopy was performed, and 2 months later an operation to resect the lung metastases via thoracoscopy was performed. Currently, 3 months after surgery, the patient is alive, without recurrence.

[A Case of Locally Advanced Rectal Cancer Successfully Treated by Conversion Surgery after Multidisciplinary Treatment].

A 70-year-old woman who complained of abdominal pain and a prolapsed tumor from the anus was diagnosed with an intestinal obstruction resulting from anal canal cancer. Computed tomography (CT) and magnetic resonance imaging revealed a huge tumor (11×5×12 cm) invading the vagina and levator ani muscle. Enlarged inguinal lymph nodes on both sides indicated metastasis. The clinical stage was T4b (vagina, levator ani muscle, and pudenda) N0H0M1a (LYM), stage IV (Japanese Classification of Colorectal Carcinoma: 8th edition). As curative resection was not possible, a transvers colostomy was performed to relieve the intestinal obstruction. This was followed by chemoradiotherapy (45 Gy/1.8 Gy×25; TS-1, 80 mg/body for 2 weeks and a 1-week interval, for 2 courses) and up to 10 courses of Bev+mFOLFOX6 continuously. After this regimen, there was a remarkable reduction in tumor size. Positron emission tomography-CT revealed no FDG uptake in the primary rectal site or inguinal lymph nodes, but a maximum standardized uptake value (SUVmax) of 6.3 was detected in the vagina. Six weeks after chemotherapy, the patient underwent a pelvic exenteration including resection of the vagina, bladder, and pudenda. The pathological stage was yp T4b (vagina) N0H0M0, stageⅡ. Curative resection was performed, and the patient had a Grade 2 pathological response after chemoradiotherapy.

A case of biliary cystic tumor with repeated hemobilia.

Intraductal papillary neoplasm of the bile duct (IPNB) is classified as a biliary cystic tumor with a tendency of causing obstruction. Neoplastic cases involving hemobilia are rarely reported. We herein describe a case of biliary cystic tumor with repeated hemobilia. A 57-year-old woman was histologically diagnosed with cavernous hemangioma. During the follow-up period after transcatheter arterial embolization (TAE), she experienced repeated hemobilia, and multiple other TAE sessions were performed for hemostasis. She was referred to our hospital 8 years after the first surgery owing to a growing tumor. Histopathological examination after extended right hepatectomy and caudate lobectomy indicated IPNB with an associated invasive carcinoma. Six months thereafter, computed tomography revealed a recurrent liver tumor and a nodule in the abdominal cavity. She died 36 months after the second surgery, despite chemotherapy. Our experience suggests that IPNB should be considered during differential diagnosis of dilated hepatobiliary tumors with hemobilia.

A case of adenoid cystic carcinoma of the esophagus.

Esophageal adenoid cystic carcinoma (EACC) is a very rare form of malignant tumor in the esophagus. Here, we report the case of a 78-year-old man who was diagnosed with EACC by preoperative endoscopic biopsy. Thoracoscopy-assisted subtotal esophagectomy with lymph node dissection was carried out. Microscopic examination of the resected specimen suggested that the tumor invaded to submucosal layer and showed no lymph node metastasis. Histologically, tumor primarily exhibited an alveolar solid pattern with partial cribriform and tubular patterns. Alcian blue staining showed many mucoid materials within the glandular cavity formed by tumor cells. Immunohistochemical studies revealed that the tumor cells reacted with pan-cytokeratin immunostains and expressed vimentin and S-100 protein. Collectively, the tumor was diagnosed as primary EACC, T1bN0M0 according to "Japanese Classification of Esophageal Cancer 10th edition." The patient showed no recurrence sign 12 months after the surgery.The current study also reviewed 35 EACC cases reported in Japanese literatures from 1990 to 2014. Combined with our case, we found that EACC is less frequently accompanied by lymph node metastasis as compared to esophageal squamous cell carcinoma, especially at the early stage. The prognosis of EACC is relatively better when tumors have no lymph node metastasis.

[A Case of Small Bowel Cancer Treated with Laparoscopic Surgery].

We report a case of small intestinal adenocarcinoma treated with laparoscopic surgery. A 70-year-old woman had abdominal pain and epigastralgia. There were no abnormal findings on upper and lower endoscopy. The symptoms continued for 4 months after endoscopy, so she presented to our hospital. After CT examination, small bowel cancer with ileus was suspected. An ileus tube was inserted to relieve the bowel pressure and she was diagnosed with ileum cancer by enteroscopy. Laparoscopic surgery was performed and the pathological stage was determined as pStage Ⅲa. She was treated with oral chemotherapy (UFT plus LV) and had no recurrence 6 months after surgery.

[Usefulness of Metallic Stent for Left-Sided Obstructive Colon Cancer].

The aim of this study was to compare the efficacy of self-expandable metallic stent (SEMS) and transanal tube as preoperative treatments for left-sided obstructive colon cancer. Forty-three patients (the SEMS group: 28 cases, the tube group: 15 cases) were included in this study. Clinicopathological data (age, sex, tumor location, depth, histological type, stage) were comparable between the 2 groups. In addition, there was no difference in intestinal decompression rate between the SEMS group and the tube group (technical success rate: 100% vs 86.7%, clinical success rate: 92.8% vs 73.3%, complication rate: 7.1% vs 0%). A significantly higher number of patients in the SEMS group underwent laparoscopic surgery because of difference of historical background. However, no significant difference was observed between the 2 groups in postoperative outcome(complication rate, hospital stay duration). SEMS insertion had several benefits compared to transanal tube placement, such as the resumption of oral intake because of rapid resolution of obstruction and easier management because SEMSs do not require washing. SEMS insertion could be a safe and effective bridge to subsequent surgery in patients with left-sided obstructive colon cancer.

[A case of clinically diagnosed eosinophilic bronchiolitis].

A 71-year-old man was referred to our hospital because of an intractable productive cough. Although he was treated for bronchial asthma, the symptom did not improve. Furthermore, since he developed progressive dyspnea and hypoxemia, he was admitted to our hospital. Marked eosinophilia in a blood test and sputum, poorly defined centrilobular nodules throughout the bilateral lung fields in a chest CT scan, and mixed ventilatory impairment in a spirometric test were revealed. Thoracoscopic lung biopsy and bronchoalveolar lavage were not conducted because of progressive respiratory failure. Therefore, we clinically diagnosed eosinophilic bronchiolitis, and immediately administered oral prednisolone (30 mg daily). His symptoms and examination findings rapidly improved. This case suggests that eosinophilic bronchiolitis should be taken into consideration for differential diagnoses of eosinophilic lung disease and obstructive lung disease, and marked eosinophilia in sputum may be one of the useful tools for diagnosis of this disease when invasive examinations are inadequate.

Structure of the trypanosome cyanide-insensitive alternative oxidase.

In addition to haem copper oxidases, all higher plants, some algae, yeasts, molds, metazoans, and pathogenic microorganisms such as Trypanosoma brucei contain an additional terminal oxidase, the cyanide-insensitive alternative oxidase (AOX). AOX is a diiron carboxylate protein that catalyzes the four-electron reduction of dioxygen to water by ubiquinol. In T. brucei, a parasite that causes human African sleeping sickness, AOX plays a critical role in the survival of the parasite in its bloodstream form. Because AOX is absent from mammals, this protein represents a unique and promising therapeutic target. Despite its bioenergetic and medical importance, however, structural features of any AOX are yet to be elucidated. Here we report crystal structures of the trypanosomal alternative oxidase in the absence and presence of ascofuranone derivatives. All structures reveal that the oxidase is a homodimer with the nonhaem diiron carboxylate active site buried within a four-helix bundle. Unusually, the active site is ligated solely by four glutamate residues in its oxidized inhibitor-free state; however, inhibitor binding induces the ligation of a histidine residue. A highly conserved Tyr220 is within 4 Å of the active site and is critical for catalytic activity. All structures also reveal that there are two hydrophobic cavities per monomer. Both inhibitors bind to one cavity within 4 Å and 5 Å of the active site and Tyr220, respectively. A second cavity interacts with the inhibitor-binding cavity at the diiron center. We suggest that both cavities bind ubiquinol and along with Tyr220 are required for the catalytic cycle for O2 reduction.

Sequence of a fusogenic herpes simplex virus, RH2, for oncolytic virotherapy.

RH2 is a novel oncolytic herpes simplex virus type 1 (HSV-1) produced by simultaneous infection with neurovirulent γ134.5 gene-deficient HSV-1 R849 derived from strain F and the spontaneously occurring, fusogenic HSV-1 HF in cell culture. The genome of RH2 was studied using Genome Sequencer FLX. RH2 comprised 149 64 bp and it was shown that the lacZ gene was inserted into the γ134.5 gene of R849. Comparison of ORFs revealed that RH2 had 100 % identity with strain F in 21/58 unique long (UL) genes (36.2%) and 1/13 unique short (US) genes (7.7%). RH2 had 100% amino acid identity with HF10 in 24/58 UL genes (41.4%) and 9/13 US genes (69.2%). Twelve genes, including UL27 (gB), US4 (gG) and UL6 (gD), had amino acid changes unique to RH2. Amino acid changes in gB occurred at positions 459 (T→A) and 817 (L→P). Other unique features were the amino acids missing in UL36 (VP1/2) and UL46 (VP11/12). Thus, RH2 is an HF10-based vector preserving the fusogenic amino acid changes of gB but lacking the γ134.5 gene. RH2 is expected to be a version of HF10 useful for the treatment of brain tumours as well as oral squamous cell carcinoma. Spontaneously occurring HSV-1 mutants may also be useful clinically, as their genome sequences can easily be determined by this genome sequencing system.

Effect of ultrasound on herpes simplex virus infection in cell culture.

BACKGROUND: Ultrasound has been shown to increase the efficiency of gene expression from retroviruses, adenoviruses and adeno-associated viruses. The effect of ultrasound to stimulate cell membrane permeabilization on infection with an oncolytic herpes simplex virus type 1 (HSV-1) was examined. RESULTS: Vero monkey kidney cells were infected with HSV-1 and exposed to 1 MHz ultrasound after an adsorption period. The number of plaques was significantly greater than that of the untreated control. A combination of ultrasound and microbubbles further increased the plaque number. Similar results were obtained using a different type of HSV-1 and oral squamous cell carcinoma (SCC) cells. The appropriate intensity, duty cycle and time of ultrasound to increase the plaque number were 0.5 W/cm², 20% duty cycle and 10 sec, respectively. Ultrasound with microbubbles at an intensity of 2.0 W/cm², at 50% duty cycle, or for 40 sec reduced cell viability. CONCLUSION: These results indicate that ultrasound promotes the entry of oncolytic HSV-1 into cells. It may be useful to enhance the efficiency of HSV-1 infection in oncolytic virotherapy.

A novel fusogenic herpes simplex virus for oncolytic virotherapy of squamous cell carcinoma.

BACKGROUND: R849 is a neurovirulent γ134.5 gene-deficient form of herpes simplex virus type 1 (HSV-1) and has LacZ genes at the deleted sites of the γ134.5 gene. HF is a spontaneously occurring, fusogenic HSV-1 strain. The purpose of this work was to generate a virus that has the syncytial character of HF, while preserving the γ134.5 gene inactivation profile of R849 virus. RESULTS: Vero cells were infected with R849 and HF simultaneously and two viruses, RH1 and RH2, expressing the LacZ gene and inducing extensive cell fusion were selected. A polymerase chain reaction (PCR)-based analysis suggested that one copy of the γ134.5 gene is lost in RH1, whereas both copies are lost in RH2, and that the γ134.5 gene is replaced by a R849-derived DNA fragment with the LacZ gene. These viruses produced larger plaques and more progeny than the parental viruses. Infection with RH2 decreased the viability of oral squamous cell carcinoma (SCC) cells most strongly. When RH2 was injected into xenografts of oral SCC in nude mice, multinucleated cells were produced and the growth of the tumors was suppressed significantly. CONCLUSION: These results indicate that novel oncolytic HSV-1 vectors can be produced with the genetic background of the oncolytic HSV-1 HF, and that RH2 is deficient in γ134.5 genes and shows extensive cytopathic effects in oral SCC cells. RH2 may be useful in oncolytic virotherapy for oral SCC.

[18F-fluorodeoxyglucose positron emission tomography (FDG-PET) negative poorly differentiated adenocarcinoma of lung with mediastinal lymph node metastases].

A 58-year-old woman was referred to our hospital for further medical examination of bilateral lung nodules on the chest computed tomography. Standardized uptake valve (SUV) max of 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) were negative value at both lung nodules, but positive value (3.4) at a pretracheal lymph node. The size of the small lung nodule of the left lower lobe (S9) was unchanged, but the lung nodule of the right upper lobe (S1) was gradually enlarged. By the biopsy of the right lung nodule, the poorly differentiated adenocarcinoma was diagnosed pathologically. The right upper lobectomy with mediastinal lymph node dissection was performed. The metastasis was pathologically determined for FDG-PET positive lymph node. The most important reason for negative FDG-PET at primary lesion was considered that the expression of glucose transporter 1 (GLUT-1) was very few. FDG-PET has become a useful tool in the diagnosis of the pulmonary cancer, but we should understand its limitation and diagnose carefully.

Effects of intracranial surgery on pineal lipid droplets, on other structures, and on melatonin secretion.

Unique effects of sham-pinealectomy [intracranial surgery (IS)] which include reduced functional activity of the adrenal gland and suppressed circadian rhythms of the adrenal medulla, and which are reversed by pinealectomy, have been reported in rodents. To clarify the mechanisms, we investigated whether or what changes occur in pineal functional activity after IS. Sixty-six male rats of normal and IS groups were used at 50 days of age. The pineal gland was first examined by quantitative electron microscopy. The Sudan III-stained lipid droplet content of the pinealocytes and plasma melatonin level were then investigated using the same animals. In IS rats, the lipid droplet content of the pinealocytes decreased in both the dark and light phases 14 days after surgery. Mean volumetric ratio of nucleus, nucleolus, and mitochondria tended to increase in IS rats. The mean plasma concentration of melatonin showed apparent day-night changes, but no significant changes because of IS, 36 h and 14 days after surgery. But in the dark phase 14 days after surgery, plasma melatonin levels showed increased dispersion of values (P < 0.04). Thus, after IS the lipid content of pinealocytes showed changes not closely related to those of plasma melatonin level. From these and other results it is speculated that IS effects are dissimilar to usual stress responses, that day-night rhythms of functional activities of the pineal and adrenal medulla are differently controlled, and that pineal gland-dependent IS effects are most probably induced by changed sensitivity/states of target mechanisms to the pineal hormone melatonin.

Enzymatic detection of precursor cell populations of preneoplastic foci positive for gamma-glutamyltranspeptidase in rat liver.

An improved staining method for gamma-glutamyltranspeptidase (GGT) was developed using Vibratome-prepared microslices. Microscopic precursor cell populations of preneoplastic foci positive for the marker enzyme were detectable sequentially in rat liver by tracing back from 5 to 1 week after carcinogen injection in a hepatocarcinogenesis model. Mirror-image comparisons of serial sections stained for GGT activity and immunocytochemically stained for GST-P (glutathione S-transferase P-form) revealed that GGT expression was confined within GST-P(+) cell populations (GST-P(+) minifoci), which are induced in the periportal area (zone 1) of the liver. GGT expression level differed from one minifocus to another, and the larger the GST-P(+) focus, the stronger was the GGT expression in it, indicating that GST-P(+)/GGT(-) phenotypes are convertible into proliferating GST-P(+)/GGT(+) ones. Our results suggest that there are at least 2 closely related precursors, GST-P(+)/GGT(-) and GST-P(+)/GGT(+) phenotypes, of preneoplastic foci in rat chemical hepatocarcinogenesis.