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Background The emergence of molecular targeted therapies (MTTs) has altered the treatment landscape of hormone receptor-positive (HR+), human epidermal growth factor 2-negative (HER2-) metastatic breast cancer (MBC). The objective of this study was to describe treatment patterns, clinical outcomes, and safety profiles among patients with HR+/HER2- MBC treated with palbociclib, abemaciclib, or everolimus in a clinical practice setting. Methods Forty-five patients with HR+/HER2- MBC were enrolled; of these, 40 received molecular targeted therapy (MTT) in ≥3rd lines and 5 received treatment in the 1st/2nd line. The results were compared with clinical trials. Results Median progression-free survival (PFS) in all patients was 5.3 months (95% confidence interval [CI] 2.8-8.4), and a similar PFS was found for patients receiving 1st/2nd line (5.5 months, 95% CI 1.8- ) and ≥ 3rd line (5.1 months, 95% CI 2.8-9.4) treatments. Eleven patients continued with the same regimen for >1 year; treatment is ongoing for 15 patients. In 23 patients (51%), everolimus was administered prior to cyclin-dependent kinase (CDK) 4/6 inhibitors. The most frequent grade 3 or higher adverse event (AE) with CDK4/6 inhibitors was neutropenia, whereas AEs ≥ grade 3 with everolimus included Pneumocystis pneumonia, sepsis, and stomatitis. Conclusions Molecular targeted therapy (MTT) was mostly used in ≥ 3rd lines, and PFS of patients receiving 1st/2nd line and ≥ 3rd line treatments was similar; however, this study included heavily treated patients and a limited number of cases. Treatment options should take into consideration the maximal benefit to the patient based on the results of clinical trials.

Background and purpose Ionising radiation causes mutations in the genomes of tumour cells and serves as a potent treatment for cancer. However, the mutation signatures in the cancer genome following ionising radiation have not been documented. Materials and methods We established an in vitro experimental system to analyse the presence of de novo mutations in the cancer genome of irradiated (60 Gy/20 fr/4 weeks) oesophageal cancer cell lines. Subsequently, we performed whole-genome, chromatin immunoprecipitation, and RNA sequencing using untreated and irradiated samples to assess the damage to the genome caused by radiation and understand the underlying mechanism. Results The irradiated cancer cells exhibited hotspots for the de novo 8502–12966 single nucleotide variants and 954–1,331 indels on the chromosome. These single nucleotide variants primarily originated from double-stranded break repair errors, as determined using mutation signature analysis. The hotspots partially overlapped with the sites of H3K9 trimethylation, which are regions characterised by a weak capacity for double-stranded break repair. Conclusion This study highlights the signature and underlying mechanism of radiation on the cancer genome.

Because of the complexity of cancer‐immune system interactions, combinations of biomarkers will be required for predicting individual patient responses to treatment and for monitoring combination strategies to overcome treatment resistance. To this end, the “immunogram” has been proposed as a comprehensive framework to capture all relevant immunological variables. Here, we developed a method to convert transcriptomic data into immunogram scores (IGS). This immunogram includes 10 molecular profiles, consisting of innate immunity, priming and activation, T cell response, interferon γ (IFNG) response, inhibitory molecules, regulatory T cells, myeloid‐derived suppressor cells (MDSCs), recognition of tumor cells, proliferation, and glycolysis. Using genes related to these 10 parameters, we applied single‐sample gene set enrichment analysis (ssGSEA) to 9417 bulk RNA‐Seq data from 9362 cancer patients with 29 different solid cancers in The Cancer Genome Atlas (TCGA). Enrichment scores were z‐score normalized (Z) for each cancer type or the entire TCGA cohort. The IGS was defined by the formula IGS = 3 + 1.5 × Z so that patients would be well distributed over a range of scores from 1 to 5. The immunograms constructed in this way for all individual patients in the entire TCGA cohort can be accessed at “The RNA‐Seq based Cancer Immunogram Web” (https://yamashige33.shinyapps.io/immunogram/)., We propose a novel scoring and visualization method for “immunogram” to assess the cancer immunity status of individual patients using the large TCGA dataset and RNA‐Seq of each patient. Immunograms for each individual will be a useful tool for precision immuno‐oncology, although their application needs to be validated in clinical trials.

First identified as a developmental gene, HOXB9 is also known to be involved in tumor biological processes, and its aberrant expression correlates with poor prognosis of various cancers. In this study, we isolated a homeodomain-less, novel HOXB9 variant (HOXB9v) from human breast cancer cell line-derived mRNA. We confirmed that the novel variant was produced from variationless HOXB9 genomic DNA. RT-PCR of mRNA isolated from clinical samples and reanalysis of publicly available RNA-seq data proved that the new transcript is frequently expressed in human breast cancer. Exogenous HOXB9v expression significantly enhanced the proliferation of breast cancer cells, and gene ontology analysis indicated that apoptotic signaling was suppressed in these cells. Considering that HOXB9v lacks key domains of homeobox proteins, its behavior could be completely different from that of the previously described variationless HOXB9. Because none of the previous studies on HOXB9 have considered the presence of HOXB9v, further research analyzing the two transcripts individually is warranted to re-evaluate the true role of HOXB9 in cancer.

Purpose Triple-negative breast cancer (TNBC) is considered a heterogeneous population and achieving a pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) is an only prognostic factor. Previously, the TP53 signature (TP53sig)-score, an expression profile of 33 genes, is reportedly predictive of the prognosis of all types of early-stage breast cancer. Herein, we analyzed whether the TP53sig-score can be subclassified in detail with only a TNBC cohort and investigated the molecular biological characteristics of the higher TP53sig-score. Methods Publicly available data from TCGA(RNA-sequence) and METABRIC (microarray) and real clinical specimens’ s expression data (NanoString Technologies) were used to explore the prognosis and molecular features of TNBC. Results The high TP53sig-score group in the present study and the cohort in METABRIC tended to have a worse prognosis than the low TP53sig-score group (p=0.583 and 0.196, respectively). In both the pCR and non-pCR groups, the high TP53sig-score patients tended to have a poor prognosis (p=0.0739). Moreover, when the NAC response and TP53sig-score were combined, the five-year breast cancer-free rate among the four groups differed significantly (p=0.043). In addition, high TP53sig-score was related to gene ontology terms, such as “cell differentiation” and “innate immune response. Notably, this group had the potential to respond favorably to immunotherapy according to the tumor immune dysfunction and exclusion model. Conclusion Combining the response to NAC and the TP53sig-score in triple-negative breast cancer was able to predict an unfavorable prognosis. Further, patients with a high TP53sig-score showed a favorable feature associated with immunotherapy

The TP53-signature is a multi-gene signature that can predict TP53 structural mutations. It has presented remarkable ability to predict the prognosis of early-stage breast cancer. However, some samples presented discordance with the signature status and structure status. We aimed to investigate whether the mRNA expression levels or copy number variation (CNV) of MDM2 and CDKN2A influence the TP53-signature-score, subtype classification, and prognosis prediction in TP53 wild-type, luminal type early-stage breast cancer samples.We selected TP53 wild-type, luminal type early-stage breast cancer samples from The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohorts. Then, we analyzed the correlation between the TP53-signature-score and mRNA expression levels or CNV of MDM2 and CDKN2A.The samples with MDM2 copy number (CN) amplification or those with CDKN2A CN deep deletion presented higher TP53-signature-score. Moreover, samples with MDM2 CN amplification or those with CDKN2A CN deep deletion had more characteristics of the luminal B type. In addition, they showed lower estrogen response early score, which correlated with response to endocrine therapy in breast cancer. However, MDM2 and CDKN2A mRNA expression did not present the same tendency. Furthermore, samples with MDM2 CN amplification or those with CDKN2A CN deep deletion had a worse prognosis in METABRIC cohort.The MDM2 or CDKN2A CNV may be useful for classifying subtypes and predicting prognosis more accurately in TP53 wild-type, luminal type early-stage breast cancer patients.

Despite previous reports on the clinical significance of plasma fibrinogen (FNG) levels as a prognostic indicator of ESCC, its underlying mechanism remains unclear. This study aimed to validate the prognostic impact of plasma FNG levels and clarify its relationship with primary tumors in patients with esophageal squamous cell carcinoma (ESCC).The prognostic impact of FNG was evaluated in patients with ESCC who underwent esophagectomy between 2000 and 2019. The RNA sequencing of the primary ESCC site, which was from pre-operative biopsy, was performed, followed by immune profile characterization using an immunogram. Those profiles were assessed via the immunohistochemical staining of tumor-associated macrophages (TAMs) and clinical response to nivolumab.Multivariate analysis identified FNG as a significant prognostic factor in ESCC. The immunogram suggested an immunosuppressive tumor environment in the high-FNG group. Immunostaining with the TAM markers CD163 and CD204, revealed that the high-FNG group had significantly higher number of TAMs compared with the low-FNG group. The immunosuppressive characteristics were clinically validated in patients with metastatic ESCC; those who had elevated FNG levels showed poor response to nivolumab.This study successfully validated the prognostic impact of plasma FNG levels in an expanded cohort with ESCC. Accordingly, our findings showed that increased plasma FNG reflects an immunosuppressive tumor microenvironment that facilitates tumor progression and poor responses to nivolumab.

The homeobox (HOX) family consists of 39 genes whose expressions are tightly controlled and coordinated within the family, during development. We performed a comprehensive analysis of this gene family in cancer settings. Gene correlation analysis was performed using breast cancer data available in The Cancer Genome Atlas (TCGA) and data from the patients admitted to our hospital. We also analyzed the data of normal breast tissue (GSE20437). We next collected gene expression and prognosis data of breast cancer patients (GSE11121, GSE7390, GSE3494, and GSE2990) and performed unsupervised hierarchal clustering by the HOX gene expression pattern and compared prognosis. We additionally performed this analysis to leukemia (available in TCGA) and sarcoma (GSE20196) data. Gene correlation analysis showed that the proximal HOX genes exhibit strong interactions and are expressed together in breast cancer, similar to the expression observed during development. However, in normal breast tissue, less interactions were observed. Breast cancer microarray meta-data classified by the HOX gene expression pattern predicted the prognosis of luminal B breast cancer patients (p = 0.016). Leukemia (p = 0.00016) and sarcoma (p = 0.018) presented similar results. The Wnt signaling pathway, one of the major upstream signals of HOX genes in development, was activated in the poor prognostic group. Interestingly, poor prognostic cancer presented stronger correlation in the gene family compared to favorable prognostic cancer. Comprehensive analysis of the HOX family demonstrated their similar roles in cancer and development, and indicated that the strong interaction of HOX genes might be specific to malignancies, especially in the case of poor prognostic cancer.

Background Soft-tissue sarcomas are a rare group of malignant tumors that usually are treated with surgical excision and radiation therapy, but recently, pazopanib, an oral tyrosine kinase inhibitor, has been used in patients with metastases who do not respond to standard chemotherapy regimens. Based on patients with advanced soft-tissue sarcomas who had received prior chemotherapy, several clinical studies have reported the survival and sensitivity (approximately 5% to 10% sensitive) of patients with soft-tissue sarcomas treated with pazopanib. Recently, next-generation sequencing (NGS) technologies have been used to provide a wide genetic information and to develop personalized medicine in cancer treatment. However, there are few reports and no genetic analyses of patients with soft-tissue sarcomas who had a complete response (CR) to pazopanib. Questions/purposes We described the clinicopathologic features of a patient with a rare, advanced soft-tissue sarcoma who achieved a CR to pazopanib treatment. Furthermore, integrative analyses using NGS and arrays were performed to elucidate characteristic alterations, including gene mutations, copy number changes, and protein expression that were associated with response to pazopanib. Additionally, functional analyses consisting of in vitro and in vivo assays were also performed to elucidate whether the identified alterations were associated with oncogenic abilities and drug responses. Methods In a sample from a 70-year-old woman with an advanced soft-tissue sarcoma treated for 1 month with 800 mg of oral pazopanib daily, CT scans demonstrated a CR to treatment. To our knowledge, there have been no patients with soft-tissue sarcomas among several clinical trials of pazopanib that have achieved a CR and therefore, our patient is considered to be extremely rare. We performed an integrative analysis including whole-exome sequencing, transcriptome sequencing, and phosphorylation profiling of receptor tyrosine kinases (RTK) using tumor samples from a patient with a CR matched to normal samples. From here on we will refer to this patient as having a CR, although a short term high-grade partial response may be more accurate. These analyses were performed using NGS and the phosphoreceptor tyrosine kinase (phospho-RTK) array. As a validation study, we also performed target sequencing using three samples from patients with long-term stable disease and two samples from patients with progressive disease who responded to pazopanib treatment. In addition, characteristic gene alterations that were identified according to the response to pazopanib in one patient with a CR, in three patients with long-term stable disease, and in 27 patients with high-grade soft-tissue sarcomas with different histologic subtypes and different responses to pazopanib were verified by quantitative real-time polymerase chain reaction. We conducted a focus formation assay to evaluate the transforming activities of these genomic alterations. Results In the patient with a CR to pazopanib, we identified several somatic mutations including Fms related receptor tyrosine kinase 1 (FLT1) p.G38S, platelet-derived growth factor receptor alpha (PDGFRA) p.T83S, and platelet-derived growth factor receptor beta (PDGFRB) exon 13 skipping. Amplification at chromosome 12q13-14 encompassing GLI family zinc finger 1 (GLI1) and cyclin-dependent kinase-4 (CDK4) was also detected. Furthermore, an elevated PDGFRB phosphorylation level was observed in the tumor. In target sequencing analyses in five patients, one of three patients with long-term stable disease had 12q13-14 amplification. The mRNA expression of GLI1, CDK4, and pazopanib targets including PDGFRA, PDGFRB, vascular endothelial growth factor receptor (VEGFR)1-3, and stem cell factor receptor (KIT) in samples from the patient with a CR, and 27 patients with high-grade soft-tissue sarcomas was verified. The expression of GLI1 was characteristically increased in the patient with a CR and in those with long-term stable disease relative to other patients with soft-tissue sarcomas. Overexpression of GLI1 showed strong transforming potential in 3T3 cells. Moreover, the overexpression of GLI1 upregulated the expression of the PDGFRB protein and promoted phosphorylation, which was dose-dependently inhibited by pazopanib. However, inhibition of GLI1-induced transformation by pazopanib was limited in the focus formation assay; therefore, mechanisms other than PDGFRB activation may contribute to transformation. Conclusions We identified several gene alterations that might be associated with a CR and long-term stable disease in patients who received pazopanib for advanced soft-tissue sarcomas. We therefore believe that this distinct molecular profile warrants further investigation to identify predictive biomarkers of the response to pazopanib. Clinical relevance Our findings identify molecular mechanisms that possibly explain the high sensitivity of soft-tissue sarcomas to pazopanib and may lead to the development of predictive biomarkers and novel therapies in patients with this and other types of soft-tissue sarcomas.

Many driver pathways for cancer cell proliferation have been reported. Driver pathway activation is often evaluated based on a single hotspot mutation such as EGFR L858R. However, because of complex intratumoral networks, the impact of a driver pathway cannot be predicted based on only a single gene mutation. Here, we developed a novel diagnostic system named the “EGFR impact score” which is based on multiplex mRNA expression profiles, which can predict the impact of the EGFR pathway in lung cancer cells and the effect of EGFR-tyrosine kinase inhibitors on malignancy. The EGFR impact score indicated robust predictive power for the prognosis of early-stage lung cancer because this score can evaluate the impact of the EGFR pathway on the tumor and genomic instability. Additionally, the molecular features of the poor prognostic group resembled those of biomarkers associated with immune checkpoint inhibitors. The EGFR impact score is a novel prognostic and therapeutic indicator for lung adenocarcinoma.

Background Application of next-generation DNA sequencing (NGS) has recently become increasingly common in the field of clinical oncology in several countries around the world. In Japan also, a system for applying NGS to routine clinical practice is gradually being established. During this process, we introduced in Japan the tumor-profiling MSK-IMPACT (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets) assay. Methods We present here our initial experience with the use of MSK-IMPACT in 68 patients selected from two institutions in Japan between June 2016 and October 2017. Results MSK-IMPACT sequencing was successful and yielded results in specimens obtained from 64 of the 68 patients, representing an overall assay success rate of 94.1%. The top three cancer types tested were endometrial cancer (17.2%), pancreatic cancer (15.6%) and colorectal cancer (12.5%). Evaluation of the clinical actionability of the genetic alterations revealed that 25.0% of patients (n = 16) harbored at least one actionable alteration. However, enrolling the patients in a genomically matched clinical trial was difficult, mainly because most clinical trials are limited to tumors arising from a specific organ/site. One patient with microsatellite instability-high status, as determined by MSK-IMPACT, was treated with pembrolizumab and showed partial response. Conclusions Although tumor profiling by NGS and administration of genomically matched therapy is a promising strategy, because of its high cost, we need to consider how we can fit it into the Japanese medical system. Towards this end, we believe that it is important to share our initial experience for furthering precision medicine in Japan.

We investigated the plasma levels of tumor-specific cell-free DNA (cfDNA) in 17 stage I-II (early) and IV (advanced) non-small cell lung cancer (NSCLC) patients who underwent radiotherapy. Digital polymerase chain reaction (PCR) and targeted sequencing showed that total and tumor-specific cfDNA levels increased in response to radiotherapy in both early- and advanced-stage NSCLC patients. We detected high copy numbers of epidermal growth factor receptor mutations (L858R and T790M) in the cfDNA samples from stage IV NSCLC patients who underwent stereotactic body radiation therapy to treat brain metastasis related to tyrosine kinase inhibitor (TKI) treatment failure. In conclusion, our study demonstrates that radiotherapy increases tumoral cfDNA levels in the plasma and shows potential to serve as an indicator for diagnosing drug-resistant tumor-related gene mutations in early-stage NSCLC patients or those undergoing molecular targeted therapy.

// Shigeo Yamaguchi 1 , Shin Takahashi 2 , Kaoru Mogushi 3 , Yuki Izumi 1 , Yumi Nozaki 1 , Tadashi Nomizu 4 , Yoichiro Kakugawa 5 , Takanori Ishida 6 , Noriaki Ohuchi 6 , Chikashi Ishioka 2 and Shunsuke Kato 1, 3 1 Department of Clinical Oncology, Juntendo University Graduated School, Tokyo 113-8421, Japan 2 Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan 3 Diagnostics and Therapeutics of Intractable Diseases, Intractable Disease Research Center, Juntendo University Graduated School, Tokyo 113-8421, Japan 4 Department of Surgery, Hoshi General Hospital, Fukushima 963-8501, Japan 5 Department of Breast Oncology, Miyagi Cancer Center Hospital, Natori 981-1293, Japan 6 Department of Breast and Endocrine Surgical Oncology, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan Correspondence to: Chikashi Ishioka, email: chikashi@tohoku.ac.jp Keywords: TP53; breast cancer; genomic instability; transcriptome; prognostic biomarker Received: October 27, 2017 Accepted: January 30, 2018 Epub: February 08, 2018 Published: March 06, 2018 ABSTRACT Purpose: TP53 signature has a robust predictive performance for prognosis in early-stage breast cancer, but the experiment that reported this relied on public microarray data and fresh-frozen samples. Before TP53 signature can be used in a clinical setting, a simple and low-cost diagnostic system using formalin-fixed paraffin-embedded (FFPE) samples is needed. New treatments based on the biological characteristics of TP53 signature are expected to follow. Experimental Design: TP53 signature was evaluated in 174 FFPE early breast cancer specimens using digital quantification via the nCounter technique (NanoString). Patients were classified as TP53 signature mutant type ( n = 64) or wild type ( n = 110). Predictive power of TP53 signature was compared with those of other gene expression signatures in 153 fresh-frozen samples of the same cohort by RNA-seq. The molecular features of TP53 signature were elucidated using TCGA omics data and RNA-seq data to explore new therapeutic strategies for patients with TP53 signature mutant type. Results: TP53 signature was a strong predictor of prognosis and was also more accurate than other gene expression signatures and independent of other clinicopathological factors. TCGA data analysis showed that risk score of TP53 signature was an index of chromosomal and genomic instability and that TP53 signature mutant type was associated with higher PD-L1 expression, variation in copy numbers, and numbers of somatic mutations. Conclusions: TP53 signature as diagnosed using the nCounter system is not only a robust predictor of prognosis but also a potential predictor of responsiveness to immune checkpoint inhibitors.

During next generation sequencing (NGS) analysis, many missense mutations were found in a well-known oncogene, many of which were variant of uncertain significance mutations. We recently treated an adult patient with pancreatoblastoma by chemotherapy. Using an NGS cancer panel, we found a previously unreported missense mutation in the 1835 codon of the adenomatous polyposis coli (APC) gene. We also found a heterogeneous mutation in the 1835 codon of the APC gene in the patient's germline by Sanger sequencing. Although this patient did not have a history of familial adenomatous polyposis, functional analysis suggested the R1835G mutant APC showed attenuated repression of Wnt/β-catenin signaling activity. This is the first report showing a novel APC missense mutation involved in the onset of adult pancreatoblastoma.

Tropomyosin-related kinase(TRK)fusion proteins are oncogenic drivers in multiple tumors in adults and children.Larotrectinib, an orally administered selective TRK inhibitor approved in the US, exhibits inhibitory activity against tumors harboring TRK fusions and is well tolerated.Here, we report the case of an 8-year-old female child with recurrence of an NTRK fusion low-grade sarcoma treated with larotrectinib monotherapy.The patient previously underwent resection of low-grade sarcoma in the right brachialis at 6 years of age, but local recurrence occurred after 16 months.As re-operation likely required amputation, larotrectinib was commenced at a dose of 100 mg BID.Complete radiographic remission was achieved after 3 months.There were no adverse events attributed to larotrectinib treatment.After dosing for 6 months, we performed local resection, confirming pathological complete remission.The drug was stopped, and the patient showed no evidence of relapse at 4 months after resection.In this case, larotrectinib was obtained using Single Patient Expanded Access under the FDA.In this paper, we also discuss the issues faced while accessing unapproved drugs in the precision medicine era in Japan.

First identified as a developmental gene, HOXB9 is also known to be involved in tumor biological processes, and its aberrant expression correlates with poor prognosis of various cancers. In this study, we isolated a homeodomain-less, novel HOXB9 variant (HOXB9v) from human breast cancer cell line-derived mRNA. We confirmed that the novel variant was produced from variationless HOXB9 genomic DNA. RT-PCR of mRNA isolated from clinical samples and reanalysis of publicly available RNA-seq data proved that the new transcript is frequently expressed in human breast cancer. Exogenous HOXB9v expression significantly enhanced the proliferation of breast cancer cells, and gene ontology analysis indicated that apoptotic signaling was suppressed in these cells. Considering that HOXB9v lacks key domains of homeobox proteins, its behavior could be completely different from that of the previously described variationless HOXB9. Because none of the previous studies on HOXB9 have considered the presence of HOXB9v, further research analyzing the two transcripts individually is warranted to re-evaluate the true role of HOXB9 in cancer.

A 56-year-old women underwent retroperitoneal leiomyosarcoma resection in December 2011. In July 2015, CT showed multiple liver metastases, and she received first-line chemotherapy treatment with doxorubicin. After 2 courses of doxorubicin, her performance status deteriorated; therefore, she was treated with pazopanib as second-line chemotherapy. PFS with pazopanib was 5 months and that with eribulin was 2.5 months. She was then treated with trabectedin as fourth-line chemotherapy from July 2016. The liver metastases reduced, and the disease was controlled for 1 year and 6 months following administration of trabectedin. We continue to treat this patient with trabectedin, and no serious side effects have been observed.

The use of antibodies against programmed cell death 1 (PD-1), which block inhibitory T cell checkpoints, is a promising new therapy for advanced malignant melanoma and NSCLC. However, patients with autoimmune diseases were excluded at the clinical trial using such immune checkpoint inhibitor, because of the possibilities to worsen an adverse event of the autoimmune disease. Thus, the efficacy and toxicity of nivolumab using such cases have not been reported yet. A 70-year-old woman with bone and duodenal metastasis of primary mucosal melanoma with complications of the rheumatoid arthritis was treated with nivolumab. After 4 weeks injection of nivolumab, bone metastasis was diminished. After receiving six courses of nivolumab therapy, she maintained a complete response for 9 months, without rheumatic exacerbation or drug-related adverse events. Establishment of the biomarker of the effect prediction of the PD-1 antibody, the adverse event prediction will be important in future.

A blocking latex agglutination test (b-LAT) developed in this study was evaluated for the detection of antibodies against chicken anemia virus (CAV) in chickens. Polystyrene latex beads were coupled with a neutralizing monoclonal antibody (mAb) to CAV (mAb-beads). When mAb-beads were mixed with antigens prepared from the lysate of MDCC-MSB1 cells infected with CAV, agglutination occurred. A short pre-incubation of CAV antigens with CAV-specific antiserum inhibited the agglutination of mAb-beads. The test results were obtained within 5min. The specificity of b-LAT was evaluated using sera from specific pathogen-free chickens and sera containing antibodies to avian influenza virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus; nonspecific agglutination and cross-reactivity with antibodies to unrelated viruses were not observed. The examination of 94 serum samples collected from commercial breeder chickens of various ages (17-63 weeks) revealed good agreement (93.6%, Kappa value=0.82) between b-LAT and a virus neutralization test, known to be most sensitive and specific in the detection of antibodies to CAV. These results indicate that b-LAT, a simple and rapid test, is a useful and reliable tool in CAV serology.

Graphical abstractDisplay Omitted Our device can operate as a Peltier device with unipolar thermoelectric materials.The new Peltier device can be fabricated with addition of a central electric line.Unipolar Peltier devices lead to use of many kinds of thermoelectric materials.The new Peltier device can give rise to temperature gradient on itself.Temperature gradient on the new Peltier device can be changed through currents. We proposed and fabricated an NN-type Peltier device composed of two small N-type Bi2Se0.37Te2.36 thermoelectric bulk materials. This structure includes an additional electric wire between the two N-type bulks. We introduce an application of the NN-type Peltier device as a stage on which a temperature difference can be induced by altering the current, targeting a rapid amplification system for deoxyribonucleic acid (a thermal cycler for the polymerase chain reaction). The currents in the two circuits differ from each other. The current dependence of the stage temperature of the NN-type Peltier device was investigated and the temperature difference on the stage was 21.4?C at a current of 24A. To analyze the device performance, the heat balance for the Peltier device composed of two N-type bulks was obtained by considering the effects of the electric resistance and thermal conductance of the central electric wire between the two N-type materials. The Seebeck coefficient, total resistance, total thermal conductance, and heat absorption were obtained by fitting to be -4.24×10-4V/K, 2.55×10-4?, 0.159W/K, and 1.13W, respectively, which were in good agreement with those estimated using literature values. Moreover, we fabricated an NN-type thermoelectric power device with a temperature difference of 70K, an open voltage of 16V, and a maximum power of 8mW at a current of 0.9A.

Background Homeobox (HOX) family consists of 39 genes which act as master regulators in embryonic development. Each of the genes is also known to play key roles in progression of breast cancer, including epithelial to mesenchymal transition, tumor angiogenesis and endocrine therapy resistance. Although there are numerous reports on individual HOX genes and cancer, none of them have comprehensively analyzed the whole gene family. Since HOX genes strongly coordinate within the family during the embryonic period, we considered that the analysis of the whole HOX family is also indispensable in breast cancer. Methods We collected 702 breast cancer data from four publicly available array datasets (GSE11121, GSE7390, GSE3494, GSE2990) and performed unsupervised hierarchal clustering into two clusters by the expression of HOX genes. We constructed model formulas for cluster prediction by dividing the samples into learning and validation groups. We used three machine learning methods: support-vector machine (SVM), neural network and Bayes. The model formulas were validated by validation samples. We also used 512 TCGA breast cancer data to calculate covariations of the genes in breast cancer. Results By the clustering of four array datasets, the DFS of the two clusters in PAM50-classified luminal B patients were statistically different (p = 0.016), and the gene ontology analysis revealed that the Wnt pathway was activated in the poor prognostic cluster. All cluster prediction models for luminal B sample achieved accuracies of over 90%. From TCGA breast cancer data, we found that HOX genes covariate the most with other HOX genes, especially within the chromosomally proximal groups. Conclusions Comprehensive analysis of the whole HOX family lead to the prediction of luminal B breast cancer prognosis. Considering that Wnt signaling controls HOX genes during the embryonic stage, we suppose a Wnt pathway activated, poor prognostic subgroup in luminal B breast cancer which can be identified by the expression of HOX genes. The cluster prediction model by machine learning was acceptable for its future adaptation in clinical settings. We also proved that HOX genes strongly covariate within the gene family in cancer, not only during the embryonic stage. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Immune checkpoint inhibitors have been getting increasing attention in the field of cancer treatment, resulting in the investigation of numerous drugs and target cancers. Clinical trials of immune checkpoint inhibitors have focused on malignant melanomas and non-small cell lung cancer;however, recently, clinical trials have been carried out for other cancers. To date, 31 phase III clinical trials have been conducted for 13 types of cancer. Recently, the results of the CheckMate025 kidney cancer and CheckMate141 head and neck cancer trials have been reported. These reports showed that nivolumab significantly enhanced overall survival in comparison to that associated with an existing second-line treatment drug. Based on these results, the approval of nivolumab for use in renal cell cancer and head and neck cancer is expected in the near future. Furthermore, the results of 20 phase III clinical trials will be submitted from 2017 to 2019, expanding the approval of immune checkpoint inhibitors. However, many issues such as biomarker searches, the evaluation of antitumor effects, and the impact on medical economy remain to be resolved. In this report, we outline clinical trial trends and the future prospects for immune checkpoint inhibitors.

We have studied a novel Peltier device having a metallic tip which is directly sandwiched between P-type and N-type thermoelectric materials. The tip can be heated and cooled directly driven by Peltier effect, resulting in high thermal response. In this study, we report the fabrication of a portable thermal cycler using a PN sandwich-structure Peltier device. The device has copper plates with minute fin to efficiently radiate heat.

We have proposed and fabricated a NN-type Peltier device. The conventional Peltier devices have a structure called π-shaped, which is required to have both P-type and N-type thermoelectric materials. On the other hand, our NN-type Peltier device proposed here is only composed of N-type (or P-type) thermoelectric materials. In this study, we fabricated an NN-type Peltier device with a T-shaped stage, and the current dependence of the stage temperature was measured both for cooling and heating.

This study is to measure accurate temperature at a point which is cooled and heated. We proposed and fabricated a new Peltier device with a coaxial thermocouple composed of constantan and copper. Current dependence of temperature of the head of the coaxial thermocouple was measured, and it was shown that in cooling operation, the difference between designated temperatures and calibrated ones was as small as within 1°C.

Internal tandem duplications (ITD) in the juxtamembrane domain of FMS-like tyrosine kinase 3 (FLT3) cause ligand-independent constitutive activation of FLT3 kinase and its downstream signaling. FLT3-ITD mutations confer poor prognosis with high relapse rates in AML patients. FLT3-targeted therapies using tyrosine kinase inhibitors (TKIs) often induce additional point mutations within the tyrosine kinase domains (FLT3-TKD mutations), most commonly found at D835 activation loop. Additional FLT3-TKD mutations that cause secondary resistance emerge in at least 20% of patients with TKI treatment. To elucidate the alterations of transcriptome signatures of FLT3-ITD and TKD double mutations in AML, we performed cap analysis of gene expression (CAGE) sequencing for 26 primary AML samples (14 with FLT3-ITD, 12 with FLT3-ITD/D835). CAGE detects and quantifies the specific transcriptional start site (TSS) transcripts, which enables high-throughput gene expression profiling and promoter usage analysis. Altered transcription of TSS in FLT3-ITD/D835 AML samples were detected by comparison with TSS in FLT3-ITD samples, and upregulation of 310 TSS and downregulation of 22 TSS were mapped (FDR < 0.05, EdgeR). Based on Gene Ontology (GO) analysis, up-regulated genes were enriched in "apoptotic process", "intracellular signal transduction" and "immune system development", including pro-survival BCL2A1 and drug resistance related S100A8 and PRKCH. To validate these transcriptional changes, we utilized isogenic paired Ba/F3 cells transfected with FLT3-ITD or FLT3-ITD/D835. CAGE detected upregulation of 1945 TSS and downregulation of 1470 TSS in FLT3-ITD/D835 compared to FLT3-ITD cells (FDR < 0.05). TSS transcriptions of Bcl-2, Prkca, NF-κB1, Myc, and Cdkn1a (p21) were upregulated in FLT3-ITD/D835 cells. GO analysis consistently highlighted higher activation of NF-κB signaling and its downstream Bcl-2 in FLT3-ITD/D835 than in FLT3-ITD cells both for primary AML samples and Ba/F3 cells. To determine a correlation between activated promoters and transcription factors in FLT3-ITD/D835 cells, we performed an unbiased search for enriched sequence motifs using HOMER software. HOMER revealed that the promoter 4 of BCL2A1 contained a common motif of transcription factor STAT6, known to associate with NF-κB and cooperatively bound to their respective promoter elements. Ingenuity Pathway Analysis also highlighted higher activation of STAT6 in FLT3-ITD/D835 AML cells compared to FLT3-ITD. Immunoblot analysis confirmed higher expression of Bcl-2, c-Myc, p27, and lower expression of Bcl-xL and Mcl-1 in FLT3-ITD/D835 Ba/F3 compared to FLT3-ITD Ba/F3 cells. FLT3-ITD and FLT3-ITD/D835 cells showed similar expression level of Bax and Bid. We found that FLT3-ITD/D835 Ba/F3 cells proliferated slower than FLT3-ITD cells (growth rate; FLT3-ITD/D835 9.8±2.4 fold, FLT3-ITD 19.8±1.2 fold, p=0.003, 48 h) with G0/G1 accumulation (FLT3-ITD/D835 61.5±10.1%, FLT3-ITD 31.3±11.2%, p Finally, we hypothesized that targeting Bcl-2 may effectively overcome the acquired resistance of FLT3-ITD/D835 cells. Venetoclax (ABT-199/GDC-0199), a clinically available Bcl-2 selective inhibitor, as single agent was not capable of eliminating FLT3-ITD AML due to altered activation of FLT3 downstream signaling and Mcl-1 upregulation. FLT3-ITD/D835 cells showed higher expression of the Bcl-2 gene and/protein and were less dependent on FLT3 signaling compared to FLT3-ITD cells (Tabe, ASH 2017). As expected, venetoclax caused more profound cell growth inhibition and apoptosis induction in FLT3-ITD/D835 Ba/F3 than in FLT3-ITD Ba/F3 cells (IC50 2.98 μM vs 13.9 μM, ED50 28.8 μM vs 173 μM, 48 h). In conclusion, we identified Bcl-2 transcriptional activation as a novel mechanism by which the acquired D835 mutation in FLT3-ITD AML cells facilitates anti-apoptotic network activation and confers TKI resistance. Bcl-2 inhibition by venetoclax represents a putative therapeutic strategy in FLT3-ITD/TDK double-mutated AML cells. Disclosures Shah: Bristol-Myers Squibb: Research Funding; ARIAD: Research Funding. Konopleva:Stemline Therapeutics: Research Funding. Andreeff:AstraZeneca: Research Funding.

FMS-like tyrosine kinase 3 with internal tandem duplication (FLT3-ITD), the most frequent mutation in acute myeloid leukemia (AML), results in the constitutive ligand-independent activation of FLT3 receptor downstream signaling. FLT3-targeted therapies using tyrosine kinase inhibitors (TKIs) demonstrate single agent activity in these patients but long-term efficacy is limited by emerging resistance in part due to acquired point mutations in the tyrosine kinase domains (TKD) of the FLT3 gene, most commonly found at D835 within the activation loop. Exportin 1 (XPO1) mediates the nucleo-cytoplasmic transport, and is overexpressed in AML cells with FLT3-ITD mutations (Kojima, Blood, 2013). We have reported that the clinically available XPO1 inhibitor selinexor effectively inhibits cell proliferation in both FLT3-ITD and FLT3-ITD/D835 mutated cells through upregulation of TP53 and blockade of c-Myc signaling (Tabe, ASH, 2017). In this study, we aimed at developing novel mechanism-based combination therapies for TKI-resistant FLT3-ITD cells with acquired TKD mutation. First we investigated the transcriptional changes associated with XPO1 and FLT3 inhibition in FLT3-ITD and FLT3-ITD/D835 mutated cells. We utilized paired isogenic FLT3-ITD or FLT3-ITD/D835Y transfected Ba/F3 cell lines and performed the cap analysis of gene expression (CAGE) that identifies and quantifies gene expression at the transcription start sites (TSS). FLT3-ITD cells are sensitive and FLT3-ITD/D835 cells are resistant to FLT3 inhibitor quizartinib. CAGE detected the shared upregulation of 3833 TSS genes induced by selinexor in FLT3-ITD and FLT3-ITD/D835 cells (FDR Based on these findings, we focused on Bcl-2 as a novel therapeutic target in FLT3-ITD/D835 cells, and investigated the anti-leukemia efficacy of combined inhibition of Bcl-2 and XPO1. We utilized the clinically available selective Bcl-2 inhibitor venetoclax and observed synergistic anti-proliferative and pro-apoptotic effects of venetoclax and selinexor in FLT3-ITD/D835 (Combination Index, CI: 0.39 and 0.45). Simultaneous inhibition of Bcl-2 by venetoclax and FLT3 by quizartinib showed no synergistic or additive effects in FLT3-ITD/D835 cells. Western blot analysis showed that the combination of venetoclax / selinexor reduced Bcl-2 and Mcl-1 in FLT3-ITD/D835 cells, and activated cleaved caspase3. In FLT3-ITD cells, the venetoclax / selinexor combination induced only small decrease of Bcl-2, did not change Mcl-1 and Bcl-xL, and increased cleaved caspase3. These changes were not observed in wt-FLT3 transfected Ba/F3 cells. Collectively, CAGE analysis of transcription start sites identified the primary mechanism underlying the synergistic activity of concomitant inhibition of Bcl-2 and XPO1 in FLT3-ITD/D835 cells as related to the regulation of Bcl-2, Mcl-1, Bcl-xL and c-Myc. These findings suggest that Bcl-2 inhibition by venetoclax combined with XPO1 inhibition by selinexor could be developed into a promising therapeutic strategy for TKI-resistant AML patients with FLT3-ITD and secondary acquired TKD mutations. Disclosures Konopleva: Stemline Therapeutics: Research Funding. Andreeff:AstraZeneca: Research Funding.

Acute myeloid leukemia (AML) cells are highly dependent on oxidative phosphorylation (OxPhos) for survival and continually adapt to fluctuations in nutrient and oxygen availability in the bone marrow (BM) microenvironment. The first-in-class OxPhos inhibitor IACS-010759 (Molina, Nat. Medicine 2018) is currently in Phase 1 clinical trial in AML. To identify biomarkers of sensitivity or resistance to OxPhos inhibition, we performed Cap Analysis of Gene Expression analysis (CAGE) in primary AML samples (11 sensitive and 3 resistant to IACS-010759) and 6 AML cell lines (OCI-AML3, MOLM13, THP-1, U937, MV4;11, HL60). CAGE identified 41 gene transcripts that were significantly higher in IACS-010759 resistant primary AML samples than in sensitive samples (fold change >3.0, FDR We next studied OxPhos inhibition under conditions mimicking BM microenvironment, by co-culturing AML cells with healthy BM-derived mesenchymal stem cells (MSC). Anti-proliferative effects of IACS-010759 were reduced by co-culture with MSC (IC50 0.01nM vs 2.1nM OCI-AML3, 65.4nM vs >10µM MOLM13). MSC co-culture increased OCR and glycoPER, and IACS-010759 decreased OCR / increased glycoPER in both cell lines. To determine the mechanism of resistance to IACS-010759 under MSC co-culture conditions, we studied the mitochondrial exchange between AML cells and MSC. We observed the bidirectional mitochondrial transfer between MSCs and AML cells. Using AML and MSC cells stably infected with mitochondria-targeted PDHA1 (GFP in AML, dsRed in MSC) to visualize the mitochondria, we found that IACS-010759 treatment facilitated transfer of AML-derived mitochondria into the adhered MSCs (24h), and MSC-derived mitochondria transferred to floating AML cells (48-72h) along with formation of tunneling nanotubes (TNTs) of AML cells, in sensitive OCI-AML3 cells (PDHA1-GFP/DsRed double positive %, control vs IACS; 9.0% vs 23.3 %). Mitochondria transfer from MSCs to AML cells was minimally observed in MOLM13 cells with elevated basal OxPhos (4.0% vs 5.0%). We next investigated IACS-010759-induced metabolic changes in AML cells interacting with bone marrow (BM) stromal cells. Capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) metabolite analysis further detected that IACS-010759 suppressed multiple anaplerotic amino acids in OCI-AML3 cells including glutamine and glutamic acid, which was reversed by MSC co-culture along with increase in intra-autophagosomal LC3-II in OCI-AML3 cells. These findings were not observed in MOLM13. Microenvironment-induced activation of glutamine metabolism and autophagy-associated amino acid metabolism could be additional compensatory mechanisms in response to OxPhos inhibition by IACS-010759 in BM microenvironment. Taken together, basal metabolic energetic capacity including elevated OxPhos and high compensatory glycolysis confer the AML-intrinsic resistance to IACS-010759, which can be reversed by simultaneous glycolysis inhibition. BM microenvironment facilitates secondary resistance to OxPhos inhibition through modulation of amino acid metabolism and direct mitochondrial transfer. Inhibition of these processes could enhance the anti-AML efficacy of OxPhos inhibition. Disclosures Andreeff: AstraZeneca: Research Funding. Konopleva:Stemline Therapeutics: Research Funding.

InSb thin films were grown by metalorganic vapor phase epitaxy using an InAs buffer layer on sapphire (0001) substrates. The stresses and strains in InSb were controlled by the thickness of the InAs buffer layer, and it was found that with decreasing compressive stress in InSb, the crystalline quality and the electrical properties improved. The thermoelectric properties of InSb were assessed and it was found that the power factor of InSb with a thickness of 5 μm reached as high as 5.8 × 10−3 W/mK2 at 600 K.

We investigated the plasma levels of tumor-specific cell-free DNA (cfDNA) in 17 stage I-II (early) and IV (advanced) non-small cell lung cancer (NSCLC) patients who underwent radiotherapy. Digital polymerase chain reaction (PCR) and targeted sequencing showed that total and tumor-specific cfDNA levels increased in response to radiotherapy in both early- and advanced-stage NSCLC patients. We detected high copy numbers of epidermal growth factor receptor mutations (L858R and T790M) in the cfDNA samples from stage IV NSCLC patients who underwent stereotactic body radiation therapy to treat brain metastasis related to tyrosine kinase inhibitor (TKI) treatment failure. In conclusion, our study demonstrates that radiotherapy increases tumoral cfDNA levels in the plasma and shows potential to serve as an indicator for diagnosing drug-resistant tumor-related gene mutations in early-stage NSCLC patients or those undergoing molecular targeted therapy.

We fabricated a Peltier knife after a point-contact-structure Peltier device. The knife tip temperature reached -12.2°C at 16A. When direction of current, the tip was heated, and reached 174.7°C at 20A. Between 180°C and -20°C, the Peltier knife operation was changed from heating to cooling in 10s and from cooling to heating in 13s. The Peltier knife will be useful in medical treatment and bioelectronics.

We characterized the electrical and thermoelectric properties of Si-doped InSb thin films into the interface. Their electrical properties were measured in the temperature range from 100K to 600K. The values of Hall mobility at room temperature were 11300cm2/Vs, 18800cm2/Vs, 17600cm2/Vs and 20100cm2/Vs for 0nm, 3nm, 170nm, and 500nm-thick Si-doped InSb layer, respectively. The value of mobility increased using Si-doping. This result shows that the electrical and thermoelectric properties were improved by Si doping into the interface. Therefore, Si doping makes a good effect on the interface between InSb layer and InAsSb buffer layer, resulting from that defects in the InSb layer near the interface decreased by Si doping.

フォトアクティブイェロープロテイン(PYP)の水素結合ネットワークを明らかにするために，筆者らはPYPの高分解能中性子結晶構造解析を行った。PYPの水素結合ネットワークには通常の水素結合に加え，二つの短距離水素結合が存在することが知られている。筆者らは，中性子結晶構造解析に必要な巨大PYP結晶(2.89×0.85×0.79mm3)を作製し，X線で0.125nm，中性子で0.15nm分解能の回折データを収集することに成功した。更に，X線，及び中性子回折データを併用した解析法を適用した結果，PYP中の942個の水素原子の内，819個の水素原子を観測することができた。得られた構造から，二つの短距離水素結合の内，発色団とY42の間の水素結合はイオン性の短距離水素結合であったのに対し，発色団とE46の間の水素結合は低障壁水素結合であることを示すことができた。脱プロトン化した発色団のカウンタイオンは，従来，プロトン化したR52と考えられていたが，今回得られた中性子構造では，R52は電気的中性状態であることがわかった。これらの観測から，蛋白質中の電荷の安定化機構に対して，新規のモデルを提唱するに至った。ここでは，PYPの光反応に対する低障壁水素結合の役割についても議論する。

A latex agglutination test (LAT) was developed for detecting antibodies against avian influenza virus. The recombinant avian influenza virus nucleoprotein expressed in Escherichia coli was purified, coupled with latex beads, and used as an antigen for the LAT. The LAT was capable of detecting anti-avian influenza virus antibodies irrespective of the avian-influenza subtype, and in most cases, the results correlated with the results of an agar gel precipitation test (AGPT). However, in comparison with the AGPT, the LAT could detect the anti-avian influenza virus antibodies for a longer period of time after the infection. The nonspecific agglutination observed in uninfected chicken sera was resolved by pretreating the sera with dried chicken-liver powder for 1 h. The LAT is easy to perform, and even after considering the time required for pretreatment of the serum, the total time required for obtaining the results is reduced in comparison to the time required in the case of the AGPT. This easy and rapid LAT is considered to be useful for monitoring avian influenza virus infection in the field.

We studied the thermoelectric and electrical properties of Si-doped InSb thin films grown by Metalorganic Chemical Vapor Deposition. Their thermoelectric properties were evaluated using power factor (Pf =α2/ρ), which is an important criterion, and a value of 10-3 W/mK2 is standard for practical use. Maximum value of power factor was 3.05×10-4 W/mK2 at 1.56×10-7mol/min for Si flow rate dependence. Power factor of wholly Si-doped InSb thin film was 5.45×10-4 W/mK2, and power factor of partially Si-doped InSb thin film was 5.45×10-4 W/mK2. Si-doping may strongly influence the interface between InSb and substrate, and defects in the InSb layer near the interface could be decreased by Si doping.

We proposed a new type Peltier device, and succeeded in fabrication of the device, which we call a point-contact-type sandwich structure (PCS). A micro-object can be cooled or heated using our fabricated device. The tip was cooled through the Peltier effect. In air, the tip temperature reached -44.7{degree sign}C at 28A. When current was reversed, the tip was heated, and the maximum tip temperature was 221.5{degree sign}C at 20A. Moreover, the minimum time constant of tip temperature was 1.7sec at 35A for cooling, and was 2.1sec at 2A for heating.For an application of PCS device, we fabricated a pen-type Peltier device. The PCS Peltier device can be useful in medical treatment and bioelectronics.

Low-barrier hydrogen bonds (LBHBs) have been proposed to play roles in protein functions, including enzymatic catalysis and proton transfer. Transient formation of LBHBs is expected to stabilize specific reaction intermediates. However, based on experimental results and theoretical considerations, arguments against the importance of LBHB in proteins have been raised. The discrepancy is caused by the absence of direct identification of the hydrogen atom position. Here, we show by high-resolution neutron crystallography of photoactive yellow protein (PYP) that a LBHB exists in a protein, even in the ground state. We identified ≈87% (819/942) of the hydrogen positions in PYP and demonstrated that the hydrogen bond between the chromophore and E46 is a LBHB. This LBHB stabilizes an isolated electric charge buried in the hydrophobic environment of the protein interior. We propose that in the excited state the fast relaxation of the LBHB into a normal hydrogen bond is the trigger for photo-signal propagation to the protein moiety. These results give insights into the novel roles of LBHBs and the mechanism of the formation of LBHBs.

A comprehensive molecular epidemiological analysis was performed on highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype derived from poultry and wild bird during 2004-2007 in Thailand. Sequence analysis followed by phylogenetic analysis was applied to all eight segments of the viruses. Viruses belonging to clades 1 and 2.3.4 in the HA phylogenetic tree have been shown to circulate in Thailand. Our analysis revealed differential evolution of the HPAI viruses among clade 1 strains. Isolates from Phichit province in 2006 resided in two distinct branches, designated 1.p1 and 1.p2. A hemagglutination inhibition test with a panel of monoclonal antibodies demonstrated a possible antigenic drift between the Phichit isolates. Involvement of free-grazing duck practice in the area was discussed as a cause of the differential evolution among the Phichit isolates. A branch, designated 1-TGWB and consisting exclusively of isolates from zoological tigers and wild birds, was evident in all phylogenetic trees constructed in the study. The branch's existence indicated that the HPAI viruses could have been maintained in the wild bird population for a certain period, although no involvement of wild birds in HPAI transmission to poultry was evident in this study.

Background Recent studies have revealed the existence of genetic diversity in swine influenza viruses (SIVs) in the world. In Thailand, there has been a little information on the molecular characteristics of the SIVs since the first isolation of viruses of H1N1 and H3N2 subtypes in the late 1970s. Our previous study demonstrated that Thai H1N1 SIVs possessed the classical swine H1 and avian-like swine N1 genes (Takemae et al., Proceedings of the Options for the Control of Influenza VI.2007;350–353). Objectives In the present study, we genetically characterized 12 SIVs including those of H1N1, H1N2 and H3N2 subtypes isolated between 2000 and 2005. Methods We determined the entire nucleotide sequences of the eight gene segments of those isolates. Results Phylogenetic analysis revealed the existence of nine distinct genotypes amongst the Thai SIVs. These genotypes arose from multiple introductions of classical swine, avian-like swine and human viruses. The existence of two distinct sublineages within classical swine H1 and NS, avian-like swine PA and M and human H3 and N2 genes of the Thai SIVs suggested that introduction of viruses of classical swine, avian-like swine and human origins occurred twice respectively into the Thai pig population. The predominance of avian-like swine genes amongst the Thai SIVs was evident. In particular, three polymerase (PB1, PB2 and PA) and matrix genes of avian-like swine origin were retained in all the Thai SIVs examined. Conclusions These observations may suggest that genes of avian-like swine lineages have some advantages to be maintained in pigs as seen in the SIVs established through multiple introductions in other regions.

At the end of May 2005, a low-pathogenicity avian influenza (LPAI) virus of subtype H5N2 was isolated for the first time from chickens in Japan. Through active and epidemiological surveillance, 5.78 million chickens on 41 farms were found to be affected and 16 H5N2 viruses were isolated. Antigenic analysis revealed antigenic similarity of these isolates. Phylogenetic analysis showed that they originated from a common ancestor and clustered with the H5N2 strains prevalent in Central America that have been circulating since 1994. Experimental infection of chickens with the index isolate (A/chicken/Ibaraki/1/05) demonstrated that this virus replicated efficiently in the respiratory tract without clinical signs, and dust-borne and/or droplet-borne transmission was considered as a possible mode of transmission. These results suggested that the H5N2 LPAI viruses isolated in Japan were highly adapted to chickens.

We report the load characteristics of miniature thermoelectric power devices using InN thin films prepared by reactive radio-frequency (RF) sputtering. The devices are composed of 25-pair and 12-pair InN-chromel films on a SiO"2 glass substrate. The open output voltage and the maximum output power were 0.11V and 2.9x10^-^8W at @DT=262K for the 25-pair device, respectively, and 0.05V and 1.3x10^-^8W at @DT=234K for the 12-pair device, respectively. The open output voltage and the maximum output power were proportional to (@DT)^n (n=1 and 2, respectively).