An atlas of combinatorial transcriptional regulation in mouse and man.

Combinatorial interactions among transcription factors are critical to directing tissue-specific gene expression. To build a global atlas of these combinations, we have screened for physical interactions among the majority of human and mouse DNA-binding transcription factors (TFs). The complete networks contain 762 human and 877 mouse interactions. Analysis of the networks reveals that highly connected TFs are broadly expressed across tissues, and that roughly half of the measured interactions are conserved between mouse and human. The data highlight the importance of TF combinations for determining cell fate, and they lead to the identification of a SMAD3/FLI1 complex expressed during development of immunity. The availability of large TF combinatorial networks in both human and mouse will provide many opportunities to study gene regulation, tissue differentiation, and mammalian evolution.

Evaluation of off-target effects of gapmer antisense oligonucleotides using human cells.

Antisense oligonucleotide (ASO) has the potential to induce off-target effects due to complementary binding between the ASO and unintended RNA with a sequence similar to the target RNA. Conventional animal studies cannot be used to assess toxicity induced by off-target effects because of differences in the genome sequence between humans and other animals. Consequently, the assessment of off-target effects with in silico analysis using a human RNA database and/or in vitro expression analysis using human cells has been proposed. Our previous study showed that the number of complementary regions of ASOs with mismatches in the human RNA sequences increases dramatically as the number of tolerated mismatches increases. However, to what extent the expression of genes with mismatches is affected by off-target effects at the cellular level is not clear. In this study, we evaluated off-target effects of gapmer ASOs, which cleave the target RNA in an RNase H-dependent manner, by introducing the ASO into human cells and performing microarray analysis. Our data indicate that gapmer ASOs induce off-target effects depending on the degree of complementarity between the ASO and off-target candidate genes. Based on our results, we also propose a scheme for the assessment of off-target effects of gapmer ASOs.

An atlas of active enhancers across human cell types and tissues.

Enhancers control the correct temporal and cell-type-specific activation of gene expression in multicellular eukaryotes. Knowing their properties, regulatory activity and targets is crucial to understand the regulation of differentiation and homeostasis. Here we use the FANTOM5 panel of samples, covering the majority of human tissues and cell types, to produce an atlas of active, in vivo-transcribed enhancers. We show that enhancers share properties with CpG-poor messenger RNA promoters but produce bidirectional, exosome-sensitive, relatively short unspliced RNAs, the generation of which is strongly related to enhancer activity. The atlas is used to compare regulatory programs between different cells at unprecedented depth, to identify disease-associated regulatory single nucleotide polymorphisms, and to classify cell-type-specific and ubiquitous enhancers. We further explore the utility of enhancer redundancy, which explains gene expression strength rather than expression patterns. The online FANTOM5 enhancer atlas represents a unique resource for studies on cell-type-specific enhancers and gene regulation.

Outcomes after decompression surgery without fusion for patients with lumbar spinal stenosis and substantial low back pain.

PURPOSE: The aim of this study was to evaluate the efficacy of decompression surgery alone for patients with intolerable low back pain. METHODS: We retrospectively identified 222 patients who underwent spinal decompression without fusion surgery who had substantial preoperative low back pain (preoperative numerical rating scale score ≥ 5). Their clinical outcomes were assessed using the numerical rating scale and the Japanese Orthopaedic Association Back Pain Evaluation Questionnaire (JOABPEQ) preoperatively and at 3 months and 1 year after surgery. RESULTS: At 3 months and 1 year after surgery compared with baseline, there was a significant improvement in the overall mean numerical rating scale scores for low back pain (baseline = 6.8, 3 months = 2.1, 1 year = 2.7), leg pain (6.8, 2.1, 2.7), and leg numbness (6.4, 2.9, 3.2) (P < 0.05). The efficacy rate assessed by JOABPEQ was 68.1% for pain-related disorders, 47.0% for lumbar spine dysfunction, 63.3% for walking ability, 48.2% for social life dysfunction, and 21.6% for psychological disorders. When patients were classified into three groups depending on their degree of leg pain (mild, moderate, and severe), there was no significant difference in the efficacy rate between the three groups. CONCLUSION: Decompression surgery can improve low back pain, regardless of the degree of preoperative leg pain, but the average score for LBP and leg pain slightly worsened between 3 months and 1 year after surgery. These slides can be retrieved under Electronic Supplementary Material.

Shared activity patterns arising at genetic susceptibility loci reveal underlying genomic and cellular architecture of human disease.

Genetic variants underlying complex traits, including disease susceptibility, are enriched within the transcriptional regulatory elements, promoters and enhancers. There is emerging evidence that regulatory elements associated with particular traits or diseases share similar patterns of transcriptional activity. Accordingly, shared transcriptional activity (coexpression) may help prioritise loci associated with a given trait, and help to identify underlying biological processes. Using cap analysis of gene expression (CAGE) profiles of promoter- and enhancer-derived RNAs across 1824 human samples, we have analysed coexpression of RNAs originating from trait-associated regulatory regions using a novel quantitative method (network density analysis; NDA). For most traits studied, phenotype-associated variants in regulatory regions were linked to tightly-coexpressed networks that are likely to share important functional characteristics. Coexpression provides a new signal, independent of phenotype association, to enable fine mapping of causative variants. The NDA coexpression approach identifies new genetic variants associated with specific traits, including an association between the regulation of the OCT1 cation transporter and genetic variants underlying circulating cholesterol levels. NDA strongly implicates particular cell types and tissues in disease pathogenesis. For example, distinct groupings of disease-associated regulatory regions implicate two distinct biological processes in the pathogenesis of ulcerative colitis; a further two separate processes are implicated in Crohn's disease. Thus, our functional analysis of genetic predisposition to disease defines new distinct disease endotypes. We predict that patients with a preponderance of susceptibility variants in each group are likely to respond differently to pharmacological therapy. Together, these findings enable a deeper biological understanding of the causal basis of complex traits.

[A Case of Metastatic Mucinous Adenocarcinoma Derived from the Right Ovary to the Ileal Ureter].

A 76-year-old woman presented with temporary right back pain and renal dysfunction. She had undergone ileal ureter interposition for right ureteral resection due to retroperitoneal liposarcoma about forty years ago. Abdominal computed tomographic (CT) scan showed right hydronephrosis and a stenosis in the middle of the ileal ureter. The symptoms were relieved, and the patient was followed. Six months later, however, a contrast enhanced lesion was newly observed at the stenosis of the ileal ureter, and nineteen months later, right ovarian tumor and peritoneal dissemination were subsequently observed. Aiming at diagnosis and cytoreduction, right nephroureterectomy and bilateral oophorectomy were performed. Histopathological findings of the resected tumors showed common characteristics of infiltrating and proliferating adenocarcinoma. Immunohistochemistry findings showed that Cytokeratin 7 and Cytokeratin 20 stained positive. On the other hand, Mucin 2 and Special AT-rich sequence-binding protein 2 were not found. The histopathological diagnosis was metastatic mucinous adenocarcinoma of the ovary with ileal ureter and a wide peritoneal dissemination. The patient rejected adjuvant chemotherapy and a new lesion was found in the pelvic fourmonths afterthe surgery.

[A Case of Malignant Lymphoma with an Elevated Serum IgG4].

A man in his 60s was referred to our hospital for further examination of left hydronephrosis and renal dysfunction. An enhanced abdominal computed tomographic scan showed that the patient had chronic abdominal aortic dissection and a non-enhancing retroperitoneal soft tissue occupying the front of the abdominal aorta as well as the bilateral common iliac arteries. The left ureter was compressed by the soft tissue at the fourth lumbar level. No tumor markers were significantly elevated and idiopathic retroperitoneal fibrosis was suspected to be the cause. Before starting treatment, however, right hydronephrosis was newly observed. We placed bilateral ureteral stents and reviewed our diagnosis. Elevated serum IgG4 and accumulation of 18F-fluorodeoxyglucose in the soft tissue were the points at issue. To determine the diagnosis, we performed open wedge biopsy. Histopathological findings showed mainly fibrous connective tissue with lymphocytic infiltration, which was positive for CD10, CD20, and bcl-2. These findings indicated follicular lymphoma. Induction chemotherapy was performed with 6 cycles of rituximab/cyclophosphamide/vincristine/prednisolone. The soft tissue tumor shrank markedly and the patient has been free from bilateral ureteral stents.

[Two-Stage Radical Resection of a Case of Colon Cancer with Abdominal Wall Invasion and Cholangiocarcinoma].

A woman in her 50s was admitted to our hospital with fever and lower abdominal swelling. Abdominal CT/MRI examinations revealed irregular thickening of the transverse colon wall, which was attached to a subcutaneous abscess. An abdominal wall mass, a patent urachus, and a tumor in the 5th segment of the liver were also noted. Colonoscopy revealed type 2 advanced transverse colon cancer. The solitary, sessile tumor was observed at the apex of the bladder under cystoscopy, suggesting the formation of the urachal carcinoma. Transcutaneous liver biopsy obtained from the liver tumor indicated adenocarcinoma, which was morphologically different from the existing transverse colon cancer. Right hemicolectomy with resection of the umbilicus, abdominal wall, urachus, and part of the bladder wall was performed. Diagnosis of the transverse colon cancer invading the abdominal wall and bladder was confirmed by histopathological examination. Hepatectomy was performed in the next surgery, and the tumor was histopathologically diagnosed as an intrahepatic cholangiocarcinoma. Both the transverse colon cancer and the intrahepatic cholangiocarcinoma were radically resected. Radical surgical diagnostic resection may be valuable in cases of multicentric cancers of unknown primary origin, if radical resection of each individual tumor is required.

[A Case Report : Metastatic Carcinoma of the Collecting Ducts of Bellini in a Hemodialysis Patient Treated with Temsirolimus].

Carcinoma of the collecting ductsof Bellini isa rare histological subtype of renal cell carcinoma and mostly has unfavorable prognosis. Radical nephrectomy is generally chosen for the 1st line treatment but therapeutic approaches for the metastasis/recurrence have not been established. We report a case of carcinoma of collecting ducts of Bellini in a patient receiving hemodialysis treated with temsirolimus. A 62- year-old man receiving hemodialysis was admitted to our hospital with drug-resistant anemia and high-grade cyclic fever. Computed tomography revealed the right renal tumor and multiple metastatic lung tumors. Open radical nephrectomy wasperformed. Pathological findingswere compatible with carcinoma of the collecting ducts of Bellini. He was given weekly temsirolimus treatment. The disease progressed modestly but kept the stable disease (SD) status for six months. He died of the cancer 11 months after the initial diagnosis.

[A Case of Triple Negative Spindle Cell Carcinoma of the Breast and Improved Quality of Life Following Irinotecan Chemotherapy].

We report a case of triple negative spindle cell carcinoma of the breast, responsive to irinotecan chemotherapy. A 49-year old woman who had a tumor in the chest wall with a skin ulcer visited our hospital. After being diagnosed with triple negative spindle cell carcinoma of the breast, she underwent surgery, adjuvant chemotherapy, and radiation at the other hospital. Fourteen months after the surgery, she developed an ipsilateral breast tumor as a result of local recurrence. Since eribulin and paclitaxel plus bevacizumab chemotherapies were not effective, she was transferred to our hospital, and we administered irinotecan as third-line chemotherapy. Skin lesions and effusion were reduced and her quality of life improved for 4 months.

[A Case of Unresectable Locally Advanced Pancreatic Cancer Resulted in R0 Surgery after Chemotherapy].

A 67-year-old man visited our hospital for jaundice. Abdominal dynamic CT showed the hypovascular tumor at the head of the pancreas that surrounded superior mesenteric artery(SMA)at an angle of 220 degree. No metastasis in lymph nodes and other organs was observed. We diagnosed the tumor unresectable locally advanced(UR-LA)pancreatic cancer. Chemotherapy was administered with gemcitabine and nab-paclitaxel(GEM+nab-PTX)and achieved partial response. Regression in size and in range around SMA to an angle of 150 was observed. We assessed it possible to resect the tumor curatively, and performed subtotal stomach preserving pancreaticoduodenectomy and dissection of the plexus around the SMA, resulted in R0 surgery. Adjuvant chemotherapy was administered, and no recurrence was observed up to present, more than a year. It is suggested that GEM+nab-PTX can be effective as the primary therapy against UR-LA pancreatic cancer.

Adsorption of RNA on mineral surfaces and mineral precipitates.

The prebiotic significance of laboratory experiments that study the interactions between oligomeric RNA and mineral species is difficult to know. Natural exemplars of specific minerals can differ widely depending on their provenance. While laboratory-generated samples of synthetic minerals can have controlled compositions, they are often viewed as "unnatural". Here, we show how trends in the interaction of RNA with natural mineral specimens, synthetic mineral specimens, and co-precipitated pairs of synthetic minerals, can make a persuasive case that the observed interactions reflect the composition of the minerals themselves, rather than their being simply examples of large molecules associating nonspecifically with large surfaces. Using this approach, we have discovered Periodic Table trends in the binding of oligomeric RNA to alkaline earth carbonate minerals and alkaline earth sulfate minerals, where those trends are the same when measured in natural and synthetic minerals. They are also validated by comparison of co-precipitated synthetic minerals. We also show differential binding of RNA to polymorphic forms of calcium carbonate, and the stabilization of bound RNA on aragonite. These have relevance to the prebiotic stabilization of RNA, where such carbonate minerals are expected to have been abundant, as they appear to be today on Mars.

The enhancer and promoter landscape of human regulatory and conventional T-cell subpopulations.

CD4(+)CD25(+)FOXP3(+) human regulatory T cells (Tregs) are essential for self-tolerance and immune homeostasis. Here, we describe the promoterome of CD4(+)CD25(high)CD45RA(+) naïve and CD4(+)CD25(high)CD45RA(-) memory Tregs and their CD25(-) conventional T-cell (Tconv) counterparts both before and after in vitro expansion by cap analysis of gene expression (CAGE) adapted to single-molecule sequencing (HeliScopeCAGE). We performed comprehensive comparative digital gene expression analyses and revealed novel transcription start sites, of which several were validated as alternative promoters of known genes. For all in vitro expanded subsets, we additionally generated global maps of poised and active enhancer elements marked by histone H3 lysine 4 monomethylation and histone H3 lysine 27 acetylation, describe their cell type-specific motif signatures, and evaluate the role of candidate transcription factors STAT5, FOXP3, RUNX1, and ETS1 in both Treg- and Tconv-specific enhancer architectures. Network analyses of gene expression data revealed additional candidate transcription factors contributing to cell type specificity and a transcription factor network in Tregs that is dominated by FOXP3 interaction partners and targets. In summary, we provide a comprehensive and easily accessible resource of gene expression and gene regulation in human Treg and Tconv subpopulations.

Novel biomarkers that assist in accurate discrimination of squamous cell carcinoma from adenocarcinoma of the lung.

BACKGROUND: Targeted therapies based on the molecular and histological features of cancer types are becoming standard practice. The most effective regimen in lung cancers is different between squamous cell carcinoma (SCC) and adenocarcinoma (AD). Therefore a precise diagnosis is crucial, but this has been difficult, particularly for poorly differentiated SCC (PDSCC) and AD without a lepidic growth component (non-lepidic AD). Biomarkers enabling a precise diagnosis are therefore urgently needed. METHODS: Cap Analysis of Gene Expression (CAGE) is a method used to quantify promoter activities across the whole genome by determining the 5' ends of capped RNA molecules with next-generation sequencing. We performed CAGE on 97 frozen tissues from surgically resected lung cancers (22 SCC and 75 AD), and confirmed the findings by immunohistochemical analysis (IHC) in an independent group (29 SCC and 45 AD). RESULTS: Using the genome-wide promoter activity profiles, we confirmed that the expression of known molecular markers used in IHC for SCC (CK5, CK6, p40 and desmoglein-3) and AD (TTF-1 and napsin A) were different between SCC and AD. We identified two novel marker candidates, SPATS2 for SCC and ST6GALNAC1 for AD, as showing comparable performance and complementary utility to the known markers in discriminating PDSCC and non-lepidic AD. We subsequently confirmed their utility at the protein level by IHC in an independent group. CONCLUSIONS: We identified two genes, SPATS2 and ST6GALNAC1, as novel complemental biomarkers discriminating SCC and AD. These findings will contribute to a more accurate diagnosis of NSCLC, which is crucial for precision medicine for lung cancer.

Scanning Electron Microscope-Cathodoluminescence Analysis of Rare-Earth Elements in Magnets.

Scanning electron microscope-cathodoluminescence (SEM-CL) analysis was performed for neodymium-iron-boron (NdFeB) and samarium-cobalt (Sm-Co) magnets to analyze the rare-earth elements present in the magnets. We examined the advantages of SEM-CL analysis over conventional analytical methods such as SEM-energy-dispersive X-ray (EDX) spectroscopy and SEM-wavelength-dispersive X-ray (WDX) spectroscopy for elemental analysis of rare-earth elements in NdFeB magnets. Luminescence spectra of chloride compounds of elements in the magnets were measured by the SEM-CL method. Chloride compounds were obtained by the dropwise addition of hydrochloric acid on the magnets followed by drying in vacuum. Neodymium, praseodymium, terbium, and dysprosium were separately detected in the NdFeB magnets, and samarium was detected in the Sm-Co magnet by the SEM-CL method. In contrast, it was difficult to distinguish terbium and dysprosium in the NdFeB magnet with a dysprosium concentration of 1.05 wt% by conventional SEM-EDX analysis. Terbium with a concentration of 0.02 wt% in an NdFeB magnet was detected by SEM-CL analysis, but not by conventional SEM-WDX analysis. SEM-CL analysis is advantageous over conventional SEM-EDX and SEM-WDX analyses for detecting trace rare-earth elements in NdFeB magnets, particularly dysprosium and terbium.

[Recurrent Gastric Carcinoma(Endocrine Cell Type)Successfully Treated with Multidisciplinary Therapy - A Case Report].

A 62-year-old woman was diagnosed with carcinoma of the stomach at another hospital. Distal gastrectomy with D2 dissection was performed and she was referred to our hospital. Histopathological and immunopathological examinations showed the tumor to be composed of adenocarcinoma and neuroendocrine carcinoma. The patient was followed until 4 months after the operation when an abdominal computed tomographic(CT)scan showed a metastatic tumor at S2 and S5/6 of the liver. No other organ metastases were found, and a hepatectomy was performed. The primary tumor of the stomach consisted of adenocarcinoma and neuroendocrine carcinoma; however, the resected metastatic liver tumor consisted of only neuroendocrine carcinoma. Liver and lung metastases appeared 2 months after the operation, and we started chemotherapy with VP-16 and cisplatin. After 8 courses of treatment, the lung metastases showed a CR, and the liver metastasis was SD. She is alive without lung metastases 9 months after the hepatectomy.

Promoter architecture of mouse olfactory receptor genes.

Odorous chemicals are detected by the mouse main olfactory epithelium (MOE) by about 1100 types of olfactory receptors (OR) expressed by olfactory sensory neurons (OSNs). Each mature OSN is thought to express only one allele of a single OR gene. Major impediments to understand the transcriptional control of OR gene expression are the lack of a proper characterization of OR transcription start sites (TSSs) and promoters, and of regulatory transcripts at OR loci. We have applied the nanoCAGE technology to profile the transcriptome and the active promoters in the MOE. nanoCAGE analysis revealed the map and architecture of promoters for 87.5% of the mouse OR genes, as well as the expression of many novel noncoding RNAs including antisense transcripts. We identified candidate transcription factors for OR gene expression and among them confirmed by chromatin immunoprecipitation the binding of TBP, EBF1 (OLF1), and MEF2A to OR promoters. Finally, we showed that a short genomic fragment flanking the major TSS of the OR gene Olfr160 (M72) can drive OSN-specific expression in transgenic mice.

A case of insulinoma with non-alcoholic fatty liver disease: Roles of hyperphagia and hyperinsulinemia in pathogenesis of the disease.

Nonalcoholic fatty liver disease (NAFLD) is a serious health-related condition all over the world; the number of patients is increasing in Asian countries including Japan. Better understanding of its pathophysiology is required to develop effective therapeutics, as patients may go on to develop non-alcoholic steatohepatitis and hepatocellular carcinomas. While NAFLD is believed to be associated with metabolic risk factors such as obesity, diabetes, and dyslipidemia, its etiology remains largely unknown and the development or co-existence of NAFLD in patients with insulinoma has not been investigated. A 33-year-old male with an insulinoma, who had been hypoglycemic during the previous four years, developed abnormally elevated levels of liver enzymes and histological fatty liver characteristic of NAFLD by the time of admission to our hospital for resection of an insulinoma. His medical records for the previous eight years revealed that his bodyweight had increased gradually from 60 kg to 71 kg for seven years and then acutely increased to 79 kg in the latest one-year period. This sudden increase was thought to be due to the patient's self-described overeating of fruits to forestall hypoglycemia. Fresh fruits are rich in fructose, and the patient's triglycerides, alanine and aspartate transaminases showed an acute increase in the previous one-year period. After resection of the insulinoma, the levels of these parameters all were mostly restored, which suggests that hyperinsulinemia and subsequent hyperphagia played a role in the development of NAFLD in this case. This is the first report of patient with NAFLD and an insulinoma.

Nonimmunoglobulin target loci of activation-induced cytidine deaminase (AID) share unique features with immunoglobulin genes.

Activation-induced cytidine deaminase (AID) is required for both somatic hypermutation and class-switch recombination in activated B cells. AID is also known to target nonimmunoglobulin genes and introduce mutations or chromosomal translocations, eventually causing tumors. To identify as-yet-unknown AID targets, we screened early AID-induced DNA breaks by using two independent genome-wide approaches. Along with known AID targets, this screen identified a set of unique genes (SNHG3, MALAT1, BCL7A, and CUX1) and confirmed that these loci accumulated mutations as frequently as Ig locus after AID activation. Moreover, these genes share three important characteristics with the Ig gene: translocations in tumors, repetitive sequences, and the epigenetic modification of chromatin by H3K4 trimethylation in the vicinity of cleavage sites.

Genome-wide analysis of mammalian promoter architecture and evolution.

Mammalian promoters can be separated into two classes, conserved TATA box-enriched promoters, which initiate at a well-defined site, and more plastic, broad and evolvable CpG-rich promoters. We have sequenced tags corresponding to several hundred thousand transcription start sites (TSSs) in the mouse and human genomes, allowing precise analysis of the sequence architecture and evolution of distinct promoter classes. Different tissues and families of genes differentially use distinct types of promoters. Our tagging methods allow quantitative analysis of promoter usage in different tissues and show that differentially regulated alternative TSSs are a common feature in protein-coding genes and commonly generate alternative N termini. Among the TSSs, we identified new start sites associated with the majority of exons and with 3' UTRs. These data permit genome-scale identification of tissue-specific promoters and analysis of the cis-acting elements associated with them.