[Practice of Ethics Education Using a Documentary Film for Pharmacy Students].

In today's world, where clinical options are ever increasing and patients' needs are more diverse, it is not possible to conclude that simply practicing medical care based on pathophysiological data and medical evidence is sufficient for patients, particularly in terms of seeing each patient as an individual. Medical professionals must maintain a close relationship with their patients and seek treatment and care methods that reflect the patient's values and views on life and death, based on their own ethics in medical care. Ethics education should be provided on a continuing basis from the beginning of medical/pharmacy school. However, ethics education in pharmacy departments is often delivered in a lecture format attended by many students and/or as group training using case studies and hypothetical situations, i.e., "paper" patients. With these teaching methods, there are limited opportunities for the students to foster a sense of ethics or to think deeply about their values and views on life and death with respect to the patients they care for. Therefore, in this study, we conducted ethics exercises for pharmacy students in a group study format using a documentary film of real patients who were facing death. By retrospectively analyzing the results of the questionnaires collected before and after the assignments and exercises, we verified the educational effects and changes in the students' sense of ethics from participating in the group learning exercise; moreover, our results revealed the insight gained by the students in examining the experiences and challenges faced by terminally ill patients.

A Complex of Type I Platelet-Activating Factor Acetylhydrolase (PAF-AH) Catalytic Subunits Switches from α1/α2 Heterodimer to α2/α2 Homodimer during Adipocyte Differentiation of 3T3-L1 Cells.

Platelet-activating factor acetylhydrolase (PAF-AH) hydrolyzes an acetyl ester at the sn-2 position of platelet-activating factor (PAF), thereby mediating a variety of biological functions. PAF-AH is found in three isoforms: Type I PAF-AH (PAF-AH I) and Type II PAF-AH (PAF-AH II) are intracellular enzymes whereas plasma PAF-AH is characterized by association with lipoprotein in plasma. PAF-AH I forms a tetramer constituted by two catalytic subunits (α1 and α2) with ß regulatory subunits. We recently showed that a deficiency of PAF-AH I catalytic subunits in male mice caused an increase of body weight, food intake, and white adipose tissue (WAT) weight. In this study, we examined whether the expression of this enzyme was altered in the differentiation of 3T3-L1 preadipocytes into adipocytes. The amount of PAF-AH I α1 subunit protein was significantly reduced in 3T3-L1 differentiation, while the amount of the PAF-AH I α2 subunit was not changed. Immunoprecipitation analysis of 3T3-L1 differentiation showed that the complex of PAF-AH I catalytic subunits was changed from α1/α2 heterodimer to α2/α2 homodimer. Our findings suggest that changes in PAF-AH I catalytic subunits are involved in adipocyte differentiation of 3T3-L1 and obesity in mice.

Genome-wide screening identified SEC61A1 as an essential factor for mycolactone-dependent apoptosis in human premonocytic THP-1 cells.

Buruli ulcer is a chronic skin disease caused by a toxic lipid mycolactone produced by Mycobacterium ulcerans, which induces local skin tissue destruction and analgesia. However, the cytotoxicity pathway induced by mycolactone remains largely unknown. Here we investigated the mycolactone-induced cell death pathway by screening host factors using a genome-scale lenti-CRISPR mutagenesis assay in human premonocytic THP-1 cells. As a result, 884 genes were identified as candidates causing mycolactone-induced cell death, among which SEC61A1, the α-subunit of the Sec61 translocon complex, was the highest scoring. CRISPR/Cas9 genome editing of SEC61A1 in THP-1 cells suppressed mycolactone-induced endoplasmic reticulum stress, especially eIF2α phosphorylation, and caspase-dependent apoptosis. Although previous studies have reported that mycolactone targets SEC61A1 based on mutation screening and structural analysis in several cell lines, we have reconfirmed that SEC61A1 is a mycolactone target by genome-wide screening in THP-1 cells. These results shed light on the cytotoxicity of mycolactone and suggest that the inhibition of mycolactone activity or SEC61A1 downstream cascades will be a novel therapeutic modality to eliminate the harmful effects of mycolactone in addition to the 8-week antibiotic regimen of rifampicin and clarithromycin.

Thyroid stimulating hormone suppresses the expression and activity of cytosolic sulfotransferase 1a1 in thyrocytes.

Sulfonation is an important step in the metabolism of dopamine, estrogens, dehydroepiandrosterone, as well as thyroid hormones. However, the regulation of cytosolic sulfotransferases in the thyroid is not well understood. In a DNA microarray analysis of rat thyroid FRTL-5 cells, we found that the mRNA expression of 10 of 48 sulfotransferases was significantly altered by thyroid stimulating hormone (TSH), with that of sulfotransferase family 1A member 1 (SULT1A1) being the most significantly affected. Real-time PCR and Western blot analyses revealed that TSH, forskolin and dibutyryl cyclic AMP significantly suppressed SULT1A1 mRNA and protein levels in a time- and concentration-dependent manner. Moreover, immunofluorescence staining of FRTL-5 cells showed that SULT1A1 is localized in the perinuclear area in the absence of TSH but is spread throughout the cytoplasm with reduced fluorescence intensity in the presence of TSH. Sulfotransferase activity in FRTL-5 cells, measured using 3'-phosphoadenosine-5'-phosphosulfate as a donner and p-nitrophenol as an acceptor substrate, was significantly reduced by TSH. These findings suggest that the expression and activity of SULT1A1 are modulated by TSH in thyrocytes.

Nobiletin and 3'-Demethyl Nobiletin Activate Brown Adipocytes upon ß-Adrenergic Stimulation.

Brown adipose tissue (BAT) specifically regulates energy expenditure via heat production. Nobiletin (NOB), a natural polymethoxylated flavone present in citrus fruits, can activate thermogenesis in the BAT of high-fat diet-induced obese mice. The activity of BAT is directly regulated by ß-adrenergic stimulation. In this study, we report the effects of NOB on BAT activation using ß-adrenergic agonists. We observed that when HB2 brown adipocyte cell lines are stimulated with ß-adrenergic agonists, NOB enhances the expression of uncoupling protein 1 (UCP1), which is associated with the mitochondrial energy metabolism in these cells. Moreover, NOB increases the mRNA expression of the brown adipokines neuregulin-4 (Nrg4) and fibroblast growth factor-21 (FGF-21) and the secretion of FGF-21. These results suggest that NOB can enhance the thermogenic functions of brown adipocytes and promote brown adipokine secretion due to enhanced ß-adrenergic stimulation. In addition, 3'-demethyl nobiletin (3'-DMN), an NOB CYP-enzyme metabolite, can increase UCP1 mRNA expression. Both NOB and 3'-DMN significantly promoted mitochondrial membrane potential in HB2 adipocytes following ß-adrenergic stimulation. Therefore, we believe that NOB could be a promising candidate for activating BAT under ß-adrenergic stimulation and preventing the onset of obesity.

Deficiency of Type I Platelet-Activating Factor-Acetylhydrolase Catalytic Subunits Causes an Increase in Body Weight.

Type I platelet-activating factor-acetylhydrolase (PAF-AH) forms a complex consisting of two catalytic subunits (α1 and/or α2) with a regulatory subunit (ß). Although this protein was discovered as an enzyme that degrades an acetyl ester linked at the sn-2 position of platelet-activating factor (PAF), its physiological function remains unknown. In this study, to examine whether knockout mice lacking the catalytic subunits of this enzyme showed a different phenotype from that of wild-type mice, we measured and compared the body weights of knockout mice and control mice. The body weights of knockout mice were significantly increased compared to those of the control mice during 6 to 20 weeks from birth. Food intake was also significantly increased in knockout mice compared with control mice during these periods. Since a decrease in testis weight was reported in the knockout mice, we expected a decrease in testosterone levels. We measured and compared the amounts of testosterone in the serum and testis of knockout and control mice using liquid chromatography-tandem mass spectrometry, and found that testosterone levels in both the serum and testis were significantly decreased in the knockout mice compared with the control mice. These results suggest that a deficiency of type I PAF-AH catalytic subunits causes an increase in body weight, in part, due to reduced testosterone levels in male mice.

[Analysis of Basic Life Support Training Provided to Pharmacy Students Using Feedback Device].

The quality of chest compression affects survival after sudden cardiac arrest, particularly when it occurs out of hospital. Pharmacy students should acquire basic life support skills as part of the model core curriculum of pharmacy education. Here, we trained first-year students at the Faculty of Pharmacy to deliver cardiopulmonary resuscitation and used a manikin with a real-time feedback device that quantified chest compression skills. Students were classified into shallow compressions (SC; <50 mm) and deep compressions (DC; ≥50 mm) groups based on the depth of chest compressions measured prior to training. After training, the mean compression depth (mm) was significantly shallower for the SC, than the DC group and many students in the SC group did not reach a depth of 50 mm. Similarly, students were classified into slow compression rate (SR; ≤120/min) and rapid compression rate (RR; >120/min) groups based on the results of training in the rate of chest compressions. Significant differences in mean compression rates were not found between the groups. However, correct compression rate (%), the percentage of maintaining 100-120 compression/min was significantly higher in the SR, than in the RR group. Chest compression rates correlated with compression depth, and chest compression tended to be too shallow in group that was too fast. The quality of chest compression might be improved by delivering chest compressions at a constant rate within the recommended range.

Epidemiological and pathological study of feline morbillivirus infection in domestic cats in Japan.

BACKGROUND: Feline morbillivirus (FmoPV) is a novel paramyxovirus found to infect domestic cats. FmoPV has been isolated in several countries in Asia and Europe and is considered to have genetic diversity. Also, it is suspected to be associated with feline renal diseases including tubulointerstitial nephritis (TIN), which affects domestic cats with a high incidence rate. RESULTS: To clarify the state of FmoPV infection among domestic cats in Japan, an epidemiological survey was conducted. Twenty-one out of 100 cats were found to have serum antibodies (Ab) against FmoPV-N protein by indirect immunofluorescence assay (IF) using FmoPV-N protein-expressing HeLa cells. Twenty-two of the cats were positive for FmoPV RNA in the urine and/or renal tissues. In total, 29 cats were positive for Ab and/or viral RNA. These FmoPV-infected cats were classified into three different phases of infection: RNA+/Ab + (14 cats), RNA+/Ab- (8 cats) and RNA-/Ab + (7 cats). In immunohistochemistry (IHC), 19 out of 29 cats were positive for FmoPV-N protein in kidney tissues; however, the FmoPV-N protein was located in the inflammatory lesions with severe grade in only four out of the 19 cats. Since 15 out of 29 infected cats were positive for viral RNA and Ab, approximately half of the infected cats were persistently infected with FmoPV. CONCLUSIONS: A statistically significant difference was observed between infection of FmoPV and the presence of inflammatory changes in renal lesions, indicating a relationship between FmoPV infection and feline renal diseases. However, we could not obtain histopathological evidence of a relationship between FmoPV infection and TIN.

Identification of a natural recombination in the F and H genes of feline morbillivirus.

Feline morbillivirus (FmoPV) has recently been identified in Hong Kong and Japan. FmoPV is considered to belong to the genus Morbillivirus, in the family Paramyxoviridae. In this study, the complete nucleotide sequences of three strains of FmoPV detected in cats in Japan were determined. Among the six genes in FmoPV; N, P/V/C, M, F, H and L, the P gene showed the highest polymorphism in the nucleotide and putative amino acid sequences among the FmoPV strains. There was no geographical association in terms of the FmoPV phylogeny; however, from extensive phylogenetic and recombination analyses, we found that one Japanese FmoPV strain, MiJP003, was a probable recombinant between two virus strains in the independent lineages found in Japan and Hong Kong, respectively. The recombination was considered to have occurred within the F and H genes. Such recombination is thought to be involved in the evolution of FmoPV.

[Base in four types of lidocaine preparation (formulated in hospital)].

PL cream (combination of lidocaine and procaine) was launched on the market in April 2012 in Japan. We investigated differences in the anesthetic effect by employing two types of base: Carbopol and methylcellulose. Electron microscopy showed a distinct difference in appearance: densely-scattered, fine particles for Carbopol and sparse, large particles for methylcellulose. Accordingly, the extensibility of the cream was significantly greater at 4 and 25 degrees centigrade for methylcellulose, but was greater at 34 degrees centigrade for Carbopol. The steady flow viscosity (1 s(-1)) was greater for the Carbopol than methylcellulose base. The difference in the cutaneous permeability between the two bases increased over time: the methylcellulose base was removed at 90 min after application and, 30 min later, showed a significant difference. These results suggest that the methylcellulose base has a superior anesthetic effect in clinical settings.

[Establishment of a practical training program in smoking cessation for use by pharmacists using cognitive-behavioral therapy and the motivational interview method].

We established a practical training program to nurture pharmacists who can give smoking cessation instructions. The program was provided to 85 interns (45 males and 40 females) in Teikyo University Hospital. The one-day practical training was provided to groups comprised of five members each. The training consisted of studies on the adverse effects of smoking, general outlines of the outpatient smoking cessation service, experiencing Smokerlyzer, studies about smoking-cessation drugs, studies about a smoking cessation therapy using cognitive-behavioral therapy and motivational interviewing, and case studies applying role-playing. Before and after the practical training, we conducted a questionnaire survey consisting of The Kano Test for Social Nicotine Dependence (KTSND) and the assessment of the smoking status, changes in attitudes to smoking, and willingness and confidence to give smoking cessation instructions. The overall KTSND score significantly dropped from 14.1±4.8 before the training to 8.9±4.8 after the training. The confidence to give smoking cessation instructions significantly increased from 3.4±1.9 to 6.2±1.3. Regarding the correlation between the smoking status and willingness and confidence to give smoking cessation instructions, the willingness and confidence were lower among the group of interns who either smoked or had smoked previously, suggesting that smoking had an adverse effect. A total of 88.2% of the interns answered that their attitudes to smoking had "changed slightly" or "changed" as a result of the training, indicating changes in their attitudes to smoking. Given the above, we believe that our newly-established smoking cessation instruction training is a useful educational tool.

Simultaneous imaging of membrane antigen and the corresponding chromosomal locus in pathology archives.

A new procedure for the simultaneous staining of membranous antigens, such as tyrosine kinase-type cell surface receptor HER2 (c-erbB2), and the corresponding chromosome (chromosome 17 for c-erbB2) in the same cell for use in examining pathology archives is presented. A multistep procedure involving microwave-assisted fluorescence in situ hybridization and immunofluorescence yielded cell images having c-erbB2 on the membrane and genomic signals from the chromosome 17 centromere and the c-erbB2 locus. Furthermore, a combination of microwave-assisted chromogenic in situ hybridization and immunohistochemistry found colorized signals from both chromosome 17 centromere in the nuclei and c-erbB2 on the membranes of individual cells. Quantitative image analysis further confirmed the presence of a significantly stronger c-erbB2 immunoreactivity on cells containing three or more signals from chromosome 17 than from those with less than three signals. It was possible to extend the constellation of cell surface markers and corresponding chromosomes or locus-specific makers to several other genes including CDH1. In this case, the disappearances of CDH1 expression, a CDH1 locus signal, and a centromere enumeration probe (CEP) 16 signal were simultaneously demonstrated in the less-adhesive tumor cells. Thus, it is believed that this procedure might pave the way for exploiting pathology archives for the genotype-phenotype analysis of individual cells.

Downregulation of EphA7 by hypermethylation in colorectal cancer.

A significant reduction of EphA7 expression in human colorectal cancers was shown using semiquantitative reverse transcription-polymerase chain reaction analysis in 59 colorectal cancer tissues, compared to corresponding normal mucosas (P=0.008), and five colon cancer cell lines. To investigate the mechanism of EphA7 downregulation in colorectal cancer, we examined the methylation status of the 5'CpG island around the translation start site in five colon cancer cell lines using restriction enzymes, methylation-specific PCR, and bisulfite sequencing and found evidence of aberrant methylation. The expression of EphA7 in colon cancer cell lines was restored after treatment with 5-aza-2'-deoxycytidine. Analysis of methylation status in totally 75 tumors compared to clinicopathological parameters revealed that hypermethylation of colorectal cancers was more frequent in male than in female (P=0.0078), and in moderately differentiated than in well-differentiated adenocarcinomas (P=0.0361). There was a tendency that hypermethylation in rectal cancers was more frequent than in colon cancers (P=0.0816). Hypermethylation was also observed in colorectal adenomas. This is the first report describing the downregulation of an Eph family gene in a solid tumor via aberrant 5'CpG island methylation. It provides the evidence that EphA7 gene is involved in human colorectal carcinogenesis.

Impaired nuclear translocation of CAR in hepatic preneoplastic lesions: association with an attenuated CYP2B induction by phenobarbital.

Phenobarbital (PB) induction of CYP2B, a representative target gene of constitutive androstane receptor (CAR), has been observed to be attenuated in preneoplastic lesions of rat liver; however, molecular basis for this attenuation is poorly understood. In this report, we provide evidence indicating that the CAR expressed in the hepatic preneoplastic lesions of rats and mice was resistant to nuclear translocation and transactivation of the PB-responsive enhancer module upon PB treatment. These observations suggest that the attenuation of the induction of CYP2B by PB in hepatic preneoplastic lesions is evidently a consequence of impaired nuclear translocation of CAR.

Correlation of EPHA2 overexpression with high microvessel count in human primary colorectal cancer.

Evidence suggests that the erythropoietin-producing hepatocellular (EPH) receptor tyrosine kinases (RTKs) and their ephrin (EFN) ligands are involved in human carcinogenesis. Expression of two of them, EFNA1 ligand and its receptor, EPHA2, has been proposed to contribute to tumor-induced neovascularization. Colorectal cancers were examined for expressions of EPHA2 and its ligand EFNA1 by semi-quantitative RT-PCR, and double-immunostained for EPHA2 and CD34. Microvessels in the tumors were counted. Double-staining was also performed in 25 cases of adenoma with focal cancer for comparison. Trends of overexpression of both EPHA2 and EFNA1 was found in tumor tissue compared to the corresponding normal tissue in the same specimen [22/37 (59.5%) and 25/37 (67.5%), respectively; P = 0.100 for EPHA2 and P = 0.009 for EFNA1]. Overexpression of EPHA2 and EFNA1 was noted more frequently in the early stage than in the late stage [EPHA2, 15/21 (71.4%) vs. 7/16 (43.8%), P = 0.007; EFNA1, 15/21 (71.4%) vs. 10/16 (62.5%), P = 0.007]. Both EPHA2 and EFNA1 were more frequently overexpressed in smaller tumors (less than 5 cm) than in larger tumors [EPHA2, 15/21 (71.4%) vs. 7/16 (43.8%), P = 0.017; EFNA1, 16/21 (76.2%) vs. 8/16 (50%), P = 0.001]. Tumors less than 5 cm in diameter and in stages I and II were significantly more likely to overexpress EPHA2 and EFNA1 (P = 0.001 for EPHA2, P = 0.001 for EFNA1). Microvessel counts (MVCs) after immunostaining for CD34 were significantly correlated (r = 0.343, P = 0.037) with overexpression of EPHA2. EPHA2-expressing focal cancer also surrounded microvessels in adenomas with focal cancers. These findings suggest an involvement of EPHA2 in colon carcinogenesis, mainly in stages I and II, and probably through their effect on microvessel induction.

Refractory ulcerative colitis complicated by a cytomegaloviral infection requiring surgery: report of a case.

Cytomegalovirus (CMV) infection has been reported to be a cause of refractory ulcerative colitis (UC). We herein report a case of refractory ulcerative colitis complicated by CMV infection requiring surgery. A 22-year-old man was admitted to our hospital with lower abdominal pain and bloody diarrhea. Under a diagnosis of acute UC, he was treated with prednisone 60 mg/day and sulfasalazine. Since his symptoms appeared to improve, the prednisone dosage was gradually reduced to 20 mg/day. After 5 months, he had an unexpected flare-up with fever and fresh anal bleeding. Colonoscopy demonstrated a punched out ulcer in the sigmoid colon. Biopsies by colonoscopy revealed cytomegalic inclusion bodies. Serologic and immunologic studies also suggested a recent CMV infection. Under a diagnosis of intractable UC complicated by a CMV infection, ganciclovir therapy was carried out, and the steroid therapy was tapered. Although the serum antigenemia became negative after the antiviral therapy, follow-up colonoscopy confirmed the severe stenosis after the punched-out ulcer healed completely. Since his symptoms did not improve, it was necessary to perform an elective proctocolectomy despite antiviral therapy. He was discharged with an uneventful postoperative course. It is important to recognize CMV colitis as a complication of inflammatory bowel disease, particularly in severe steroid-resistant colitis. Furthermore, in cases which fail to respond to antiviral treatment, the patient may ultimately require surgery.

Duodenal cancer in a patient with Peutz-Jeghers syndrome: molecular analysis.

We experienced an unusual case of duodenal adenocarcinoma associated with Peutz-Jeghers syndrome (PJS). A 34-year-old woman was admitted to our hospital with abdominal pain. She had been diagnosed as having PJS at 21 years of age, based on the presence of mucocutaneous pigmentation of the lip and fingertips, and colonic hamartomatous polyps. Abdominal computed tomography revealed a tumor in the third portion of the duodenum extending into the pancreas head. As the tumor was pathologically determined to be adenocarcinoma at the time of surgery, pylorus-preserving pancreaticoduodenectomy was performed. We carried out molecular analyses of this patient to examine the pathway of carcinogenesis in PJS. The tumor did not show somatic mutation of the APC and K-ras genes, which is a critical step for the adenoma-carcinoma sequence in colon cancer. Importantly, a germline mutation of the STK11 gene was detected at codon 281 delC in exon 6. Moreover, the tumor showed loss of heterozygosity of the 19p marker near STK11 and somatic mutation of the p53 gene. These findings suggest that STK11 is a tumor suppressor gene regulating the development of hamartomas, and that somatic mutation of p53 subsequently promotes gastrointestinal cancer at a later stage in PJS.

Diverticulitis causing a high serum level of carbohydrate antigen 19-9: report of a case.

We report herein a rare case of diverticulitis causing a high serum level of carbohydrate antigen (CA) 19-9. A 52-year-old man was admitted to our hospital with lower abdominal pain. Laboratory data showed evidence of inflammation and a high serum level of CA 19-9 (370 U/ml). Computed tomography demonstrated thickening of the wall of the sigmoid colon. He was diagnosed as having diverticulitis of the sigmoid colon and was treated with antibiotics. Although his symptoms improved, the presence of a malignancy such as colorectal cancer could not be completely ruled out because of the persistently high serum level of CA 19-9. A laparotomy was performed and the sigmoid colon was found to be adherent to the bladder. Under a diagnosis of diverticulitis, a sigmoidectomy was performed. Pathological examination revealed diverticulitis of the sigmoid colon, but there was no evidence of malignancy in the resected specimen. The serum CA 19-9 level decreased to normal postoperatively and immunohistochemical staining revealed CA 19-9 antigen in the cytoplasm of the diverticular epithelium. Therefore, a possible explanation for the high level of this tumor marker was diverticulitis of the sigmoid colon.

Aggressive angiomyxoma of the perineum originating from the rectal wall.

Aggressive angiomyxoma (AA) is a rare mesenchymal tumor that preferentially involves the pelvic and perineal regions, and is characterized by frequent local recurrences. We describe here a case of large AA in a 31-year-old woman. The patient was admitted to our hospital with a mass in the perineal region, associated with severe menstrual pain. Although her past medical history was unremarkable, she had spotty pigmentation on the lips. Magnetic resonance imaging showed a large mass in the abdominal pelvis traversing the pelvic diaphragm just to the right of the anus, and the border between the tumor and the rectal wall was indistinct. Pathology examination of a frozen intraoperative specimen suggested AA, and, therefore, we completely resected the tumor, using a combined abdominoperineal approach. The tumor was attached to the right wall of the rectum and the pelvic diaphragm between the anus and the puborectalis. The patient recovered uneventfully and there has not been any evidence of local recurrence for 3 years postoperatively. We consider that abdominoperineal resection may be an appropriate treatment for a large AA infiltrating to the perirectal tissues, because the high recurrence rate of this disease has been attributed to incomplete surgical excision.