Synthesis and Field-Effect Transistor Application of π-Extended Lactam-Fused Conjugated Oligomers obtained by Tandem Direct Arylation.

Five π-extended lactam-fused conjugated oligomers (5FO, 5FS, 4FPO, 4FPS, and R-4FPO) were synthesized by the tandem direct arylation. The intermolecular oxidative direct arylation was applied in the second step. These conjugated oligomers had fine-tuned FMO energies predictable by the theoretical calculation and excellent thermal stabilities. 4FPO and 4FPS bearing tetrafluoropyridine exhibited lower LUMO energy levels (-3.20 eV and -3.39 eV, respectively) compared with others. Based on the X-ray crystallography, 4FPO was found to have a herringbone crystal packing and a considerably large electron transfer integral value (137 meV). 4FPO-based bottom-gate, bottom-contact FET device demonstrated an electron mobility of 5.2×10-3  cm2 V-1 s-1 as a result of an edge-on alignment on the SiO2 substrate.

[A Case of Ischemic Colitis Four Months after Laparoscopic Left Hemicolectomy Preserving Superior Rectal Artery].

The case was for a male at the age of 80. We performed laparoscopic left hemicolectomy and D3 lymph node dissection for descending colon cancer. He had a good postoperative prognosis and was discharged on the 14th day after the operation. Later, he was receiving the treatment on an outpatient basis without postoperative adjuvant chemotherapy during the followup period. He visited the hospital for sudden abdominal pain and melena as chief complaint approximately 4 months after the operation. We found prominent edematous wall thickening and increased surrounding fat concentration in the anal side of colon from the anastomosis site with plain abdominal CT scan. We also found that the anal side of colon from the anastomosis site an edematous change broadly in the lower gastrointestinal endoscopy. We conducted conservative treatment with the diagnosis of ischemic colitis at the anal side of colon from the anastomosis site. He was discharged on the 11th day after the hospitalization. Later, we conducted a follow-up examination for him on an outpatient basis. We recognized the symptom improvement approximately 2 months after the onset of the ischemic colitis.

[Impact of Muscle Mass Reduction on Postoperative Adjuvant Chemotherapy and Long-Term Prognosis in Patients with Gastric Cancer].

BACKGROUND: Measuring the area of the psoas muscle on computed tomography is useful for the evaluation of skeletal muscle mass. The skeletal muscle is thought to be involved in weight loss after gastric surgery, and weight loss causes a decrease in compliance with chemotherapy continuity. PATIENTS AND METHODS: The psoas muscle index(PMI)was determined in 33 patients undergoing surgery for Stage Ⅱ-ⅢB gastric cancer. The rate of change in PMIwas calculated, and patients were classified into maintained and reduced muscle groups using a cutoff of -0.23 month-1. Relationships between the rate of PMIchanges and prognosis and chemotherapy continuity were examined. RESULTS: The rate of PMIchanges was significantly associated with recurrence-free survival in univariate(maintained vs reduced muscle: p=0.002)and multivariate(p= 0.0018)analyses. A reduction in the muscle mass was associated with dropout from adjuvant chemotherapy and was a predictor of a poor prognosis. CONCLUSION: The rate of PMIchanges is related to the period of adjuvant chemotherapy and is an independent prognostic factor after surgery for StageⅡ-ⅢB gastric cancer.

[A Case of Cancerous Meningitis in Juvenile Onset Gastric Cancer].

A 30-year-old woman was diagnosed with advanced gastric cancer(MUL, Circ, Type 4, por1+2, T4a, N3a, M1[LYM, P1, CY1, H0], Stage Ⅳ)on delivery. Because of unresectable, she underwent chemotherapy(first-line: S-1 plus CDDP, secondline: PTX plus Rmab, and third-line: Nmab); approximately 10 months later, she started complaining of headache. We performed a close examination, because she also developed resistance to chemotherapy. Contrast-enhanced magnetic resonance imaging of the brain revealed intense and diffuse enhancement on the brain surface, leading to the suspicion of meningeal carcinomatosis. However, hydrocephalus did not occur. She was given steroids to alleviate symptoms, but this treatment did not effective. We used neither intrathecal chemotherapy nor radiation therapy. Her symptoms gradually worsened, and she died approximately 4 weeks after the diagnosis of meningeal carcinomatosis. Meningeal carcinomatosis resulting from gastric cancer is very rare and is often difficult to diagnose. Even though this type of disease is diagnosed correctly, rapid disease progression makes the treatment difficult; therefore, patients with this type of disease have a terribly poor prognosis in daily clinical practice.

[A Study of 15 Patients with Colorectal Cancer Perforation, Kyoto Chubu Medical Center].

In colorectal cancer perforation, selecting the appropriate surgical operation while considering the patient's life and radical treatment is important. We divided 15 patients who underwent surgical intervention at our department into 2 groups, namely, free and covering perforation groups, and conducted a retrospective analysis. In the comparison between the 2 groups (free vs covering), there were 11 vs 4 cases with similar morphology, 2 vs 0 cases of perioperative death, and 3 vs 0 cases of recurrence, respectively. For the 2 groups(free vs covering), the SOFA score was 1.72 vs 1.0, postoperative chemotherapy enforcement rate was 55%vs 75%, start time was 59.4 days vs 40.3 days, and postoperative PMX implementation was 6 vs 0, respectively. All cases of recurrence and perioperative deaths were from the free perforation group. In free perforation, patients have a high risk of sepsis before surgery, and postoperative chemotherapy cannot be performed smoothly and completed. This leads to an increase in the relapse rate. It is important to select the appropriate operative method for curability and to perform postoperative chemotherapy without delay, especially in covering perforation.

Inefficient viral replication of bovine leukemia virus induced by spontaneous deletion mutation in the G4 gene.

Enzootic bovine leucosis is caused by bovine leukemia virus (BLV) infection, which is highly prevalent in several regions of the world and significantly impacts the livestock industry. In BLV infection, the proviral load in the blood reflects disease progression. Although the BLV genome is highly conserved among retroviruses, genetic variation has been reported. However, the relationship between proviral load and genetic variation is poorly understood. In this study, we investigated the changes in proviral load in BLV-infected cattle in Japan and then identified and analysed a BLV strain pvAF967 that had a static proviral load. First, examining the proviral load in the aleukaemic cattle in 2014 and 2015, cow AF967 showed a static proviral load, while the other cows showed significant increases in proviral load. Sequencing the provirus in cow AF967 showed a deletion of 12 nt located in the G4 gene. An in vitro assay system using BLV molecular clone was set up to evaluate viral replication and production. In this in vitro assay, the deletion mutation in the G4 gene resulted in a significant decrease in viral replication and production. In addition, we showed that the deletion mutation did not affect the viral transcriptional activity of Tax protein, which is also important for virus replication. The emergence of strain pvAF967 that showed a static proviral load, combined with other retrovirus evolutionary traits, suggests that some BLV strains may have evolved to be symbiotic with cattle.

SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all hemagglutinin and neuraminidase genes of avian influenza viruses and comparison to standard serological subtyping tests.

Continuing outbreaks of H5N1 highly pathogenic (HP) avian influenza virus (AIV) infections of wild birds and poultry worldwide emphasize the need for global surveillance of wild birds. To support the future surveillance activities, we developed a SYBR green-based, real-time reverse transcriptase PCR (rRT-PCR) for detecting nucleoprotein (NP) genes and subtyping 16 hemagglutinin (HA) and 9 neuraminidase (NA) genes simultaneously. Primers were improved by focusing on Eurasian or North American lineage genes; the number of mixed-base positions per primer was set to five or fewer, and the concentration of each primer set was optimized empirically. Also, 30 cycles of amplification of 1:10 dilutions of cDNAs from cultured viruses effectively reduced minor cross- or nonspecific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected, with average sensitivities of NP, HA, and NA genes of 10(1.5), 10(2.3), and 10(3.1) 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with that of current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA genes and 99% of the NA genes were genetically subtyped, while only 45% of HA genes and 74% of NA genes were serologically subtyped. Additionally, direct subtyping of AIVs in fecal samples was possible by 40 cycles of amplification: approximately 70% of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples.

Emergence of avian influenza viruses with enhanced transcription activity by a single amino acid substitution in the nucleoprotein during replication in chicken brains.

To explore the genetic basis of the pathogenesis and adaptation of avian influenza viruses (AIVs) to chickens, the A/duck/Yokohama/aq10/2003 (H5N1) (DkYK10) virus was passaged five times in the brains of chickens. The brain-passaged DkYK10-B5 caused quick death of chickens through rapid and efficient replication in tissues, accompanied by severe apoptosis. Genome sequence comparison of two viruses identified a single amino acid substitution at position 109 in NP from isoleucine to threonine (NP (I)109(T)). By analyzing viruses constructed by the reverse-genetic method, we established that the NP (I)109(T) substitution also contributed to increased viral replication and polymerase activity in chicken embryo fibroblasts, but not in duck embryo fibroblasts. Real-time RT-PCR analysis demonstrated that the NP (I)109(T) substitution enhances mRNA synthesis quickly and then cRNA and viral RNA (vRNA) synthesis slowly. Next, to determine the mechanism underlying the appearance of the NP (I)109(T) substitution during passages, four H5N1 highly pathogenic AIVs (HPAIVs) were passaged in the lungs and brains of chicken embryos. Single-nucleotide polymorphism analysis, together with a database search, suggests that the NP (I)109(T) mutation would be induced frequently during replication of HPAIVs in brains, but not in lungs. These results demonstrate that the amino acid at position 109 in NP enhances viral RNA synthesis and the pathogenicity of highly pathogenic avian influenza viruses in chickens and that the NP mutation emerges quickly during replication of the viruses in chicken brains.

NP body domain and PB2 contribute to increased virulence of H5N1 highly pathogenic avian influenza viruses in chickens.

The molecular basis of pathogenicity of H5N1 highly pathogenic avian influenza (HPAI) viruses in chickens remains largely unknown. H5N1 A/chicken/Yamaguchi/7/2004 virus (CkYM7) replicates rapidly in macrophages and vascular endothelial cells in chickens, causing sudden death without fever or gross lesions, while H5N1 A/duck/Yokohama/aq10/2003 virus (DkYK10) induces high fever, severe gross lesions, and a prolonged time to death, despite the 98% amino acid identity between the two viruses. To explore the molecular basis of this difference in pathogenicity, a series of eight single-gene reassortant viruses from these HPAI viruses were compared for pathogenicity in chickens. Two reassortants possessing the NP or PB2 gene from DkYK10 in the CkYM7 background reduced pathogenicity compared to other reassortants or CkYM7. Inversely, reassortants possessing the NP or PB2 gene of CkYM7 in the DkYK10 background (rgDkYK-PB2(Ck), rgDkYK-NP(Ck)) replicated quickly and reached higher titers than DkYK10, accompanied by more rapid and frequent apoptosis of macrophages. The rgDkYK-NP(Ck) and rgDkYK-PB2(Ck) reassortants also replicated more rapidly in chicken embryo fibroblasts (CEFs) than did rgDkYK10, but replication of these viruses was similar to that of CkYM7 and DkYK10 in duck embryo fibroblasts. A comparison of pathogenicities of seven rgDkYK10 mutants with a single amino acid substitution in NP(Dk) demonstrated that valine at position 105 in the NP(Ck) was responsible for the increased pathogenicity in chickens. NP(Ck), NP(105V), and PB2(Ck) enhanced the polymerase activity of DkYK10 in CEFs. These results indicate that both NP and PB2 contribute to the high pathogenicity of the H5N1 HPAI viruses in chickens, and valine at position 105 of NP may be one of the determinants for adaptation of avian influenza viruses from ducks to chickens.

Broad detection and quick differentiation of bovine viral diarrhea viruses 1 and 2 by a reverse transcription loop-mediated isothermal amplification test.

For broad detection of pestivirus A (bovine viral diarrhea virus 1: BVDV1) and pestivirus B (BVDV2) by a reverse transcription loop-mediated isothermal amplification (RT-LAMP) test, the P25 primer set was designed using nucleotide sequences of 5'-UTR region of 1454 BVDVs. The base coverage of each primer against diverse BVDVs were more than 99% in each base position. The one step LAMP test with the P25 primer set could detect both BVDV1 (TK) and BVDV2 (KZ), but did not amplify 5 other bovine viruses. Detection limit of the LAMP test was 103 copies of synthesized DNAs, and 10-3 and 10-4 dilutions of viral RNAs of TK and KZ strains, respectively, whereas that with current Aebischer's primer set was 10-2 dilution and negative of these RNAs, respectively. All of the 63 viral RNA samples of persistently infected (PI) cattle, consisting of the 1a (12), 1b (31), 1c (11), and 2a (9) subgenotypes, were broadly detected with the P25, while only 65% of them were positive with Aebischer's primer set. The validation study showed that the RT-LAMP test with the P25 had 100% sensitivity and 100% specificity against that with updated Vilcek's PCR primers. Also, by using the P26 primer set which contained 3 species-specific primers, all 63 RNA samples were clearly distinguished from BVDV1 or BVDV2 by the typing RT-LAMP test. These results indicate that the one step RT-LAMP test using P25 or P26 primer sets would be useful for broad detection and rapid differentiation of BVDV1 and BVDV2.

Parathyroid carcinoma detected within the thyroid.

Parathyroid carcinoma (PC) is a rare disease accounting for approximately 1% of primary hyperparathyroidism cases. The preoperative differentiation of PC is critical because PC can occasionally metastasise and invade the local tissue. However, this is challenging in asymptomatic cases and when the tumour is adjacent to the thyroid. Herein, we report a rare case of PC without clinical symptoms. Fine needle aspiration was performed, despite being contraindicated in PC, and an intrathyroidal tumour was preoperatively suggested.

Specific antiviral effect of violaceoid E on bovine leukemia virus.

Bovine leukemia virus (BLV) infection has spread worldwide causing significant economic losses in the livestock industry. In countries with a high prevalence of BLV, minimizing economic losses is challenging; thus, research into various countermeasures is important for improving BLV control. Because anti-BLV drugs have not been developed, the present study explored a promising chemical compound with anti-BLV activity. Initially, screening of a chemical compound library revealed that violaceoid E (vioE), which is isolated from fungus, showed antiviral activity. Further analysis demonstrated that the antiviral effect of vioE inhibited transcriptional activation of BLV. Cellular thermal shift assay and pulldown assays provided evidence for a direct interaction between vioE and the viral transactivator protein, Tax. These data indicate that interference with Tax-dependent transcription could be a novel target for development of anti-BLV drugs. Therefore, it is suggested that vioE is a novel antiviral compound against BLV.

Phenotypes influencing the transmissibility of highly pathogenic avian influenza viruses in chickens.

To identify critical phenotypes that affect avian influenza virus transmission in chickens, we compared the transmissibility of three H5N1 highly pathogenic viruses of different pathogenicity in chickens by monitoring the exact time of death using wireless thermo-sensors. This study showed that, despite quick deaths, the most virulent H5N1 A/chicken/Yamaguchi/7/2004 transmitted quickly in chickens via contact and airborne routes. Intermediate virulent H5N1 A/chicken/Miyazaki/K11/2007 spread moderately and less virulent H5N1 A/duck/Yokohama/aq10/2003, causing severe clinical signs and a long period to death, spread slowly among the animals. The transmissibility was correlated with virus titres of oropharyngeal and cloacal swabs, and the time for swab virus titres to reach 50 % chicken infective dose affected the transmission speed. These results demonstrate that peak virus titres excreted and the time required for virus titres to reach a minimal chicken infectious dose may be the critical phenotypes influencing the transmissibility of highly pathogenicity avian influenza viruses in chickens.

Broad detection of diverse H5 and H7 hemagglutinin genes of avian influenza viruses by real-time reverse transcription-PCR using primer and probe sets containing mixed bases.

Real-time reverse transcription-PCR (RT-PCR) was developed for broad detection of diverse H5 and H7 genes in Eurasian and American lineages of avian influenza viruses by using primer and probe sets containing mixed bases. Optimal use of the mixed bases enabled us to minimize sequence mismatches and to broaden the gene detection spectrum without decreasing sensitivity.

Association of increased pathogenicity of Asian H5N1 highly pathogenic avian influenza viruses in chickens with highly efficient viral replication accompanied by early destruction of innate immune responses.

The Asian H5N1 highly pathogenic avian influenza (HPAI) viruses have been increasing in pathogenicity in diverse avian species since 1996 and are now widespread in Asian, European, and African countries. To better understand the basis of the increased pathogenicity of recent Asian H5N1 HPAI viruses in chickens, we compared the fevers and mean death times (MDTs) of chickens infected with the Asian H5N1 A/chicken/Yamaguchi/7/04 (CkYM7) strain with those infected with the H5N1 Duck/Yokohama/aq10/03 (DkYK10) strain, using a wireless thermosensor. Asian H5N1 CkYM7 caused peracute death in chickens before fever could be induced, whereas DkYK10 virus induced high fevers and had a long MDT. Real-time PCR analyses of cytokine mRNA expressions showed that CkYM7 quickly induced antiviral and proinflammatory cytokine mRNA expressions at 24 h postinfection (hpi) that suddenly decreased at 32 hpi. In contrast, these cytokine mRNA expressions increased at 24 hpi in the DkYK10 group, but decreased from 48 hpi onward to levels similar to those resulting from infection with the low-pathogenicity H5N2 A/chicken/Ibaraki/1/2004 strain. Sequential titrations of viruses in lungs, spleens, and kidneys demonstrated that CkYM7 replicated rapidly and efficiently in infected chickens and that the viral titers were more than twofold higher than those of DkYK10. CkYM7 preferentially and efficiently replicated in macrophages and vascular endothelial cells, while DkYK10 grew moderately in macrophages. These results indicate that the increased pathogenicity in chickens of the recent Asian H5N1 HPAI viruses may be associated with extremely rapid and high replication of the virus in macrophages and vascular endothelial cells, which resulted in disruption of the thermoregulation system and innate immune responses.

Development of multipurpose recombinant reporter bovine leukemia virus.

Bovine leukemia virus (BLV) is a global problem that results in significant economic losses to the livestock industry. We developed three virus strains by inserting the HiBiT reporter tag from NanoLuc luciferase (NLuc) into limited sites within BLV molecular clones. Initial analysis for site selection of the tag insertion revealed a permissible site immediately downstream of the viral envelope gene. Therefore, NLuc activity could be used to measure virus copy numbers in the supernatant and the levels of cell infection. Productivity and growth kinetics of the reporter virus were similar to those of the wild-type strain; therefore, the reporter virus can be used to characterize the replication of chimeric viruses as well as responses to the antiviral drug, amprenavir. Collectively, our results suggest that the BLV reporter virus with a HiBiT tag insertion is a highly versatile system for various purposes such as evaluating virus replication and antiviral drugs.

Use of reverse transcriptase PCR to subtype N1 to N9 neuraminidase genes of avian influenza viruses.

Reverse transcriptase PCR designed to amplify the N1 to N9 neuraminidase (NA) genes of avian influenza viruses detected 118 of the 119 NA genes tested (99.2%) in a subtype-specific manner. This technique successfully subtyped all 167 recent avian influenza viruses isolated from birds. Subtype specificity was confirmed by sequence analyses of all 285 PCR products.

New Micro-amount of Virion Enrichment Technique (MiVET) to detect influenza A virus in the duck faeces.

Transboundary animal diseases, including highly pathogenic avian influenza, cause vast economic losses throughout the world. While it is important to identify the sources and propagation routes of the spread, such strategies are often hindered by incomplete epidemiological evidence. Isolation/detection of micro-amounts of pathogens from environmental samples is rarely successful due to the very low contamination level. This paper describes the development of the micro-amount of virion enrichment technique (MiVET), a simple and highly sensitive method that combines the use of a complex comprising a polyclonal antibody and protein G-coated magnetic beads for virion capture, and simple sodium dodecyl benzenesulfonate (SDBS) elution for low volume samples. The performance of the MiVET was evaluated using avian influenza A viruses (AIVs) in artificially spiked samples by real-time reverse transcription polymerase chain reaction (rRT-PCR). Four AIVs, H3N2, H4N2, H5N2 and H7N7, were used to artificially spike 50 ml of phosphate-buffered saline (PBS) and 1 ml of 10%-25% duck faecal supernatants. The MiVET system successfully concentrated AIVs in both PBS and faecal samples with at least 2 and 1 log greater efficacy, respectively, than conventional RNA extraction methods. The MiVET could be completed in <30 min from the beginning of sample preparation to final RNA extraction. The MiVET effectively prevented the effects of inhibitors in faecal samples, and did not require special equipment. This is the first report of this novel type of system, which is expected to be useful for the detection of micro-amounts of various veterinary and human viruses to elucidate their circulation dynamics in the environment, and for rapid and sensitive diagnosis with greater detection power.

A point mutation to the long terminal repeat of bovine leukemia virus related to viral productivity and transmissibility.

It is important to establish the molecular basis of the high transmissibility of bovine leukemia virus (BLV) to develop new methods of preventing viral transmission. Hence, the aim of this study was to determine whether some strains had transmission advantages. First, we determined the whole BLV genome sequences of all 34 BLV-infected cows from one farm. Phylogenetic analysis divided strains into 26 major and 8 minor strains. The major strains dominantly spread independent of host factor, bovine leucocyte antigen. Further analysis, with molecular clones, associated transmissibility with viral productivity in vitro. In addition, the two groups could be classified by group-specific mutations. The reverse genetic approach demonstrated that a spontaneous mutation at nucleotide 175 of the BLV genome, which is located in the viral promoter region, could alter viral productivity by changing viral transactivation, suggesting that BLV transmissibility is affected by a spontaneous mutation associated with viral productivity.

Highly pathogenic avian influenza virus (H5N1) isolated from whooper swans, Japan.

On April 21, 2008, four whooper swans were found dead at Lake Towada, Akita prefecture, Japan. Highly pathogenic avian influenza virus of the H5N1 subtype was isolated from specimens of the affected birds. The hemagglutinin (HA) gene of the isolate belongs to clade 2.3.2 in the HA phylogenetic tree.