Highly selective inhibition of histone demethylases by de novo macrocyclic peptides.

The JmjC histone demethylases (KDMs) are linked to tumour cell proliferation and are current cancer targets; however, very few highly selective inhibitors for these are available. Here we report cyclic peptide inhibitors of the KDM4A-C with selectivity over other KDMs/2OG oxygenases, including closely related KDM4D/E isoforms. Crystal structures and biochemical analyses of one of the inhibitors (CP2) with KDM4A reveals that CP2 binds differently to, but competes with, histone substrates in the active site. Substitution of the active site binding arginine of CP2 to N-ɛ-trimethyl-lysine or methylated arginine results in cyclic peptide substrates, indicating that KDM4s may act on non-histone substrates. Targeted modifications to CP2 based on crystallographic and mass spectrometry analyses results in variants with greater proteolytic robustness. Peptide dosing in cells manifests KDM4A target stabilization. Although further development is required to optimize cellular activity, the results reveal the feasibility of highly selective non-metal chelating, substrate-competitive inhibitors of the JmjC KDMs.

Effect of obstructive jaundice on hepatic hemodynamics: use of Sonazoid-enhanced ultrasonography in a prospective study of the blood flow balance between the hepatic portal vein and hepatic artery.

PURPOSE: To prospectively clarify the effects of obstructive jaundice (OJ) on hepatic hemodynamics using contrast-enhanced ultrasonography (US). METHODS: Subjects comprised 14 patients admitted to our hospital for OJ between April 2013 and March 2014. Contrast-enhanced US was performed using the LOGIQ E9 ultrasound device during the jaundice phase, before biliary drainage, and again after improvement of jaundice. After injecting the Sonazoid contrast agent, contrast dynamics were recorded in the right kidney and liver segments 5 or 6. Prototype software was used to calculate mean arrival time (AT) of the contrast agent in the liver parenchyma. Statistical analysis was performed to compare the mean AT in the jaundice and improved jaundice phases. RESULTS: We were unable to follow up three of the 14 patients after biliary drainage; thus, we included 11 patients for further analysis. The mean AT of the contrast agent was 2.0 ± 1.8 and 6.1 ± 2.3 s in the jaundice and improved jaundice phases, respectively, showing significantly shorter AT in the jaundice phase (p = 0.0033). CONCLUSION: Our findings indicate that OJ may influence the blood flow balance between the hepatic portal vein and hepatic artery.

Spontaneous complete necrosis of hepatocellular carcinoma: A case report and review of the literature.

The present study reports the case of a 68-year-old male patient who presented to Tokyo Rosai Hospital for the treatment of alcoholic liver disease. A high density was observed in liver segment S2, while a tumor, 30 mm in size, exhibiting a low density was observed in the delayed phase upon contrast-enhanced computed tomography (CT), which was performed prior to admission. The tumor appeared slightly poorly defined upon abdominal ultrasound and was observed as a 30 mm low-echoic nodule that was internally heterogeneous. A 5-mm thick contrast enhancement effect was observed in the tumor border in the vascular phase on Sonazoid contrast-enhanced ultrasonography, while a defect in the entire tumor was observed in the post-vascular phase. Dysphagia had commenced three months prior to presentation and a weight loss of ~3 kg was observed. Therefore, the patient was admitted to Tokyo Rosai Hospital due to the presence of a hepatic tumor, and to undergo a close inspection of the cause of the tumor. Upon close inspection, it was determined that the weight loss and aphagia were caused by progressive bulbar paralysis. A contrast-enhanced CT was performed on post-admission day 29 as a follow-up regarding the hepatic tumor. As a result, although no change in the tumor size was observed, the contrast enhancement in the tumor borderline had disappeared. Necrosis of the tumor was considered. However, as viable persistence of the malignant tumor could not be excluded, a hepatic left lobe excision was performed. The patient was diagnosed with hepatocellular carcinoma (HCC) based on the morphology of the cellular necrosis. In addition, occlusion due to thrombus was observed within the blood vessels passing inside the fibrous capsule. It was hypothesized that the formation of a thick fibrous capsule and occlusion due to thrombus in the feeding vessel were possibly involved as the cause of complete spontaneous necrosis. Written informed consent was obtained from the patient.

Gallstone Ileus following Endoscopic Stone Extraction.

An 85-year-old woman was an outpatient treated at Tokyo Rosai Hospital for cirrhosis caused by hepatitis B. She had previously been diagnosed as having common bile duct stones, for which she underwent endoscopic retrograde cholangiopancreatography (ERCP). However, as stone removal was unsuccessful, a plastic stent was placed after endoscopic sphincterotomy. In October 2012, the stent was replaced endoscopically because she developed cholangitis due to stent occlusion. Seven days later, we performed ERCP to treat recurring cholangitis. During the procedure, the stone was successfully removed by a balloon catheter when cleaning the common bile duct. The next day, the patient developed abdominal pain, abdominal distension, and nausea and was diagnosed as having gallstone ileus based on abdominal computed tomography (CT) and abdominal ultrasonography findings of an incarcerated stone in the terminal ileum. Although colonoscopy was performed after inserting an ileus tube, no stone was visible. Subsequent CT imaging verified the disappearance of the incarcerated stone from the ileum, suggesting that the stone had been evacuated naturally via the transanal route. Although it is extremely rare for gallstone ileus to develop as a complication of ERCP, physicians should be aware of gallstone ileus and follow patients carefully, especially after removing huge stones.

Mucinous cystadenocarcinoma of the appendix in which contrast-enhanced ultrasonography was useful for assessing blood flow in a focal nodular lesion in the tumor cavity: A case report.

A 63-year-old woman was admitted to hospital with pain in the right lower quadrant. Abdominal computed tomography (CT) revealed a 60-mm cystic mass at a site corresponding to the appendix. The mass wall on the appendicular ostium was thickened and enhanced by contrast, while calcification was observed in the mass wall on the appendicular tip. No projection was observed in the mass cavity. On abdominal ultrasonography (US), the mass wall on the appendicular ostium was thickened and projections were observed at two sites in the mass cavity. On contrast-enhanced US (CEUS), only one of these projections was enhanced. Based on the thickened and contrast-enhanced wall of the mass on the appendicular ostium on CT and US, as well as the contrast enhancement of a projection on US, the mass was diagnosed as mucinous cystadenocarcinoma of the appendix. Ileocecal resection was subsequently performed on day 10. A detailed examination of the surgical specimen revealed carcinoma cells in the mass wall on the appendicular ostium. The contrast-enhanced projection was identified as granulation tissue that had grown to come into contact with the tumor, while the non-contrast-enhanced projection was identified as solidified mucus. US enabled successful visualization of projections in the mass cavity that were not visible on abdominal CT. CEUS also proved useful for assessing blood flow in these projections.

Endoscopic nasobiliary drainage improves jaundice attack symptoms in benign recurrent intrahepatic cholestasis: A case report.

A 66-year-old male with unbearable pruritus and jaundice was admitted for detailed examination. Blood tests on admission showed increased bilirubin with a dominant direct fraction. Ultrasonography and computed tomography performed subsequent to admission showed no narrowing or distension of the bile ducts. As the jaundice symptoms were not improved by the oral administration of ursodeoxycholic acid (300 mg/day) that had been started immediately after admission, endoscopic retrograde cholangiopancreatography (ERCP) was performed on hospital day 14. This also showed no abnormalities of the bile ducts. After considerating its potential effects for improving jaundice, endoscopic nasobiliary drainage (ENBD) was performed on the same day and was followed by immediate improvements in pruritus and jaundice. Detailed examinations were performed to identify the cause of the jaundice, which was suspected to be viral hepatitis, autoimmune hepatitis or drug-induced liver injury, however, there were no findings suggestive of any of these conditions. Following a further increase in bilirubin levels, confirmed by additional blood tests, a liver biopsy was performed. Histological findings were consistent with the histological features of benign recurrent intrahepatic cholestasis (BRIC). Although ursodeoxycholic acid is used as a first-line treatment in most cases of BRIC, ENBD should also be considered for patients not responding to this treatment.

A case of branch duct type intraductal papillary neoplasm of the bile duct treated by open surgery after 11 years of follow-up.

The intraductal papillary neoplasm of the bile duct (IPNB) is a novel disease concept that was recently classified as a biliary cystic tumor by the revised World Health Organization classification. This is the case report of a 70-year-old female patient who experienced repeated episodes of obstructive jaundice and cholangitis since 2000, attributed to a mucus-producing hepatic tumor. Surgery was advised due to the repeated episodes; however, the patient refused. In May, 2011, the patient developed jaundice and fever and was treated with antibiotics. Since there was no improvement, the patient was admitted to the Tokyo Rosai Hospital. Abdominal computed tomography (CT) revealed a 50-mm cystic mass with an internal septum in the left hepatic lobe. Although the tumor size had remained almost unchanged compared to the initial CT scan performed in 2000, intra- and extra-hepatic bile duct dilation was more prominent on the second CT scan. Following admission, endoscopic retrograde cholangiopancreatography was performed and revealed an expanded papilla of Vater due to a mucous plug. A balloon catheter was inserted into the bile duct to remove the mucous plug, resulting in the drainage of copious amounts of mucus and infected bile. The patient finally consented to surgery and left hepatic lobectomy was performed. Consequently, the diagnosis of low-grade IPNB was made. Branch duct type IPNB, which is characterized by imaging appearance of a cystic mass and slow progression, is attracting increasing attention. In the present case, a cystic mass was identified in the left hepatic lobe, with no significant change in size after 11 years of follow-up, leading to the diagnosis of branch duct type IPNB. Considering the fact that IPNB is usually treated surgically at the time of diagnosis, the present case, due to the long-term follow-up, provides valuable insight into the natural history of the tumor.

Mucin phenotypes and macroscopic shape of multiple hyperplastic polyps together with two carcinomas in the stomach.

The patient was a 57-year-old Japanese woman who had been identified as having anemia and hypoproteinemia by a local group medical check-up. Esophagogastroduodenoscopy revealed an elevated lesion of 35mm in diameter on the posterior wall of the gastric antrum, surrounded by multiple polyps. The elevated lesion was diagnosed as an adenocarcinoma on the basis of biopsy, and total gastrectomy was performed. Histological examination of entire resected stomach revealed two lesions of intramucosal carcinoma together with multiple hyperplastic polyps. To investigate the mucin phenotypes and carcinogenesis of these lesions, immunohistochemical analyses of MUC2, MUC5AC, MUC6, CD10, Ki-67 and p53 protein expressions were performed in 17 hyperplastic polyps and two cancerous lesions. Expression of the MUC6 positive glands beneath the surface foveolar epithelium of hyperplastic polyps caused a morphological change from sessile to pedunculated, suggesting that this was also involved with cancerous changes. The case reported herein seems to be extremely interesting in terms of elucidating the process whereby gastric cancer arises from hyperplastic polyps.

[A case of ascariasis in the gallbladder successfully treated with an anthelmintic injection into the gallbladder via a percutaneous transhepatic catheter].

A man in his 40s who had made frequent visits abroad was admitted to our hospital complaining of epigastric pain. Ultrasonography (US) revealed an "inner tube sign" in the gallbladder, which suggested a diagnosis of ascariasis in the gallbladder. Pyrantel pamoate was directly injected into the gallbladder via a percutaneous transhepatic catheter. The worm was dead 10 minutes after the injection. US revealed reduction of the worm's length and then the disappearance of the worm from the gallbladder at both 13 days and 2 months after the injection. This method is less invasive than operation and therefore is possibly more safe. It is known that the number of cases of ascariasis may increase in Japan due to increased organic vegetable consumption and foreign travel. We need to consider this disease in the differential diagnosis of epigastric pain.

Identification of beta2-microgloblin as a candidate for early diagnosis of imaging-invisible hepatocellular carcinoma in patient with liver cirrhosis.

Glypican-3 (GPC3) is overexpressed in hepatocellular carcinoma (HCC) but not in chronic hepatitis (CH) and liver cirrhosis (LC). We have reported the possibility of GPC3-specific cytotoxic T lymphocytes (CTLs) serving as a marker for the early diagnosis of imaging invisible HCC. In this study, to identify new early diagnostic biomarker of imaging invisible HCC, we analyzed plasma of healthy donors and patients with CH, LC and HCC using surface-enhanced laser desorption-ionaization time-of-flight mass spectrometry (SELDI-TOF-MS). The intensities of four peaks were significantly increased in HCC patients compared with healthy donors. Two of these four peaks were significantly higher in CH and LC patients with GPC3-specific CTLs than in those without. One peak (11.7 kDa) was predicted to be beta2-microglobulin (beta2-MG) by molecular mass. There was a correlation between concentration of beta2-MG by latex agglutination immunoassay in plasma and peak intensity using SELDI-TOF-MS. The 11.7 kDa protein was fractionated by gel filtration and was identified as beta2-MG by Western blot analysis. These results suggest that the level of beta2-MG in plasma from patients with CH and LC could be a useful marker for the early diagnosis of imaging invisible HCC, however further investigation is needed.

Detection of glypican-3-specific CTLs in chronic hepatitis and liver cirrhosis.

Glypican-3 (GPC3) is one of carcinoembryonic antigens known to be overexpressed in hepatocellular carcinoma (HCC). It has been suggested that GPC3 may be related to the development of HCC in a background of chronic hepatitis (CH) and liver cirrhosis (LC). Therefore, in an attempt to establish an early diagnostic marker of HCC, we quantified the number of GPC3-specific CTLs in the peripheral blood of CH and LC patients. We selected CH and LC patients who were HCV-RNA (+) or HBs antigen (+) within 6 months prior to the study and had no HCC nodules as detected by imaging. A total of 56 patients with CH and LC, and 45 patients with HLA-A24+ or HLA-A2+ were enrolled for this investigation. After isolation of mononuclear cells from each patient's peripheral blood specimens, we performed ELISPOT assay using HLA-A24- and HLA-A2-restricted GPC3 peptides. In the ELISPOT assay, GPC3-specific CTLs were detected in 10 of the 45 CH and LC cases (22%). In addition, the plasma titers of anti-GPC3 IgG were increased in the CH and LC patients as compared with those in healthy donors. GPC3-specific CTLs were found to be present not only in patients with HCC, but also in patients with CH and LC. This suggests the possibility of GPC3-specific CTLs serving as a marker for the early diagnosis of imaging-invisible HCC.

Multiple vertebral wedge osteotomy for adolescent idiopathic scoliosis.

19 idiopathic and 1 syringomyelia scoliosis patients (17 females and 3 males) underwent fusionless, multiple vertebral wedge osteotomy and with follow-up for an average of 8.9 years. The average age at surgery was 16.4 years. The average curve magnitude measured by Cobb was 64.0 degrees before surgery, 38.8 degrees after surgery, 43.7 degrees one year after surgery, 46.9 degrees two years after surgery and 48.2 degrees at the latest follow-up. The difference between the Cobb angle two years after surgery and latest follow-up was not significant. There were no major complications such as neurological problems.

Fusionless surgery for scoliosis: 2-17 year radiographic and clinical follow-up.

STUDY DESIGN: Descriptive case series. OBJECTIVE: To determine whether fusionless, multiple vertebral wedge osteotomy can safely obtain correction of the deformity with adolescent idiopathic scoliosis (AIS). SUMMARY OF BACKGROUND DATA: To our knowledge, no such attempts to manage the AIS with fusionless, vertebral osteotomies have been reported. METHODS: A total of 20 consecutive patients were treated since 1987. Deformity correction, complications, respiratory function, and patient-oriented outcome were investigated. RESULTS: There were 20 patients (17 females and 3 males), including 19 with idiopathic and 1 with syringomyelia scoliosis, who underwent surgery at an average age of 16.4 years and were followed for 8.9 years (range 2-17) on average. There were no neurologic complications. One superficial wound infection necessitated debridement. There were 2 patients converted to posterior instrumentation surgery because of deterioration of the deformity. The average Cobb angle of 64.0 degrees before surgery was corrected to 48.2 degrees at 8.9 years after surgery. Decline of the pulmonary function test after surgery was not statistically significant. The patients' responses to questions about function and pain were favorable. CONCLUSION: Deformity with AIS was safely corrected with fusionless, multiple vertebral wedge osteotomy.

Two-photon microscopic analysis of acetylcholine-induced mucus secretion in guinea pig nasal glands.

The spatiotemporal changes in intracellular free Ca(2+) concentration ([Ca(2+)](i)) as well as fluid secretion and exocytosis induced by acetylcholine (ACh) in intact acini of guinea pig nasal glands were investigated by two-photon excitation imaging. Cross-sectional images of acini loaded with the fluorescent Ca(2+) indicator fura-2 revealed that the ACh-evoked increase in [Ca(2+)](i) was immediate and spread from the apical region (the secretory pole) of acinar cells to the basal region. Immersion of acini in a solution containing a fluorescent polar tracer, sulforhodamine B (SRB), revealed that fluid secretion, detected as a rapid disappearance of SRB fluorescence from the extracellular space, occurred exclusively in the luminal region and was accompanied by a reduction in acinar cell volume. Individual exocytic events were also visualized with SRB as the formation of Omega-shaped profiles at the apical membrane. In contrast to the rapidity of fluid secretion, exocytosis of secretory granules occurred with a delay of approximately 70s relative to the increase in [Ca(2+)](i). Exocytic events also occurred deep within the cytoplasm in a sequential manner with the latency of secondary exocytosis being greatly reduced compared with that of primary exocytosis. The delay in sequential compound exocytosis relative to fluid secretion may be important for release of the viscous contents of secretory granules into the nasal cavity.

Stabilization of exocytosis by dynamic F-actin coating of zymogen granules in pancreatic acini.

Reorganization of F-actin in the apical region of mouse pancreatic acinar cells during Ca(2+)-dependent exocytosis of zymogen granules was investigated by two-photon excitation microscopy with intact acini. Granules were rapidly coated with F-actin in response to either agonist stimulation or photolysis of a caged-Ca(2+) compound. Such F-actin coating occurred exclusively at the surface of granules undergoing exocytosis and was prevented either by latrunculin-A, which inhibits actin polymerization, or by Clostridium botulinum exoenzyme C3, which inhibits the small GTPase Rho. Latrunculin-A or exoenzyme C3 also triggered the formation of vacuoles in acinar cells, a characteristic of acute pancreatitis. Stimulation of acini with high concentrations of cholecystokinin, which cause acute pancreatitis in mice, also impaired the F-actin coating of granules and induced vacuole formation. Latrunculin-A reduced the latency to exocytosis but did not affect the total number of exocytic events, suggesting that F-actin slows and further stabilizes exocytosis by facilitating F-actin coating. Rho-dependent F-actin coating of granule membranes thus stabilizes exocytic structures and is necessary for physiological progression of sequetial compound exocytosis in the exocrine pancreas and for prevention of acute pancreatitis.

Sequential exocytosis of insulin granules is associated with redistribution of SNAP25.

We have investigated sequential exocytosis in beta cells of intact pancreatic islets with the use of two-photon excitation imaging of a polar fluorescent tracer, sulforhodamine B, and a fusion protein comprising enhanced cyan fluorescent protein (ECFP) and the SNARE protein SNAP25 (synaptosome-associated protein of 25 kD) transfected with an adenoviral vector. Sequential exocytosis was found to account for <10% of exocytic events in beta cells stimulated either with glucose under various conditions or by photolysis of a caged-Ca2+ compound. Multigranular exocytosis, in which granule-to-granule fusion occurs before exocytosis, was rarely found. We detected redistribution of ECFP-SNAP25 from the plasma membrane into the membrane of the fused granule occurred in a large proportion (54%) of sequential exocytic events but in only a small fraction (5%) of solitary fusion events. Removal of cholesterol in the plasma membrane by methyl-beta-cyclodextrin facilitated both redistribution of ECFP-SNAP25 and sequential exocytosis by threefold. These observations support the hypothesis that SNAP25 is a plasma membrane factor that is responsible for sequential exocytosis.

Spinal wedge osteotomy by a single posterior approach for correction of severe and rigid kyphosis or kyphoscoliosis.

STUDY DESIGN: Seven patients with severe angular kyphotic deformity of the spine were treated by circumferential spinal wedge osteotomy using a single posterior approach. OBJECTIVE: To evaluate the surgical outcomes for seven patients with severe angular kyphosis or kyphoscoliosis treated by spinal wedge osteotomy. SUMMARY OF BACKGROUND DATA: Excellent surgical outcomes have been reported for procedures such as hemivertebra excision, vertebral body resection, and spinal osteotomy for angular kyphosis or kyphoscoliosis. However, the safety and efficacy of these procedures for severe and rigid deformities have not been established. METHODS: The surgical procedure involves circumferential exposure of the apex vertebra to the anterior aspect using a single posterior approach. Sparing only the spinal cord, the surgeon performs circumferential wedge bone resection, closure, correction, and stabilization by instrumentation while monitoring the spinal cord. Seven patients (3 kyphotics and 4 kyphoscoliotics) treated by this procedure were evaluated for a minimum of 2 years. Underlying conditions comprised five cases of congenital deformity with hemivertebrae and two cases of skeletal dysplasias. The mean age at surgery was 16.5 years, and the mean follow-up period was 6.9 years. RESULTS: Before surgery, the mean kyphotic curve was 105.4 degrees (range, 68-150 degrees ), and the mean scoliotic curve was 85.3 degrees (range, 60-132 degrees ). After surgery, the curves averaged 48.9 degrees and 40 degrees, respectively, yielding corrections of 52.9% and 54.9%. The mean posterior trunk shift in global sagittal balance was 21 mm before surgery, becoming 3 mm after surgery. Progressive spinal cord dysfunction had developed in three patients before surgery. In all three, postoperative neurologic improvement was observed. Postoperative complications consisted of transient, unilateral leg paresis in two patients. No incidents of infection or pseudarthrosis were observed. CONCLUSIONS: Spinal wedge osteotomy by the single posterior approach is a reliable and safe surgical technique for correcting severe rigid angular kyphosis or kyphoscoliosis.