Keio University Hospital

The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly ‘housekeeping’, whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research. A study from the FANTOM consortium using single-molecule cDNA sequencing of transcription start sites and their usage in human and mouse primary cells, cell lines and tissues reveals insights into the specificity and diversity of transcription patterns across different mammalian cell types. FANTOM5 (standing for functional annotation of the mammalian genome 5) is the fifth major stage of a major international collaboration that aims to dissect the transcriptional regulatory networks that define every human cell type. Two Articles in this issue of Nature present some of the project's latest results. The first paper uses the FANTOM5 panel of tissue and primary cell samples to define an atlas of active, in vivo bidirectionally transcribed enhancers across the human body. These authors show that bidirectional capped RNAs are a signature feature of active enhancers and identify more than 40,000 enhancer candidates from over 800 human cell and tissue samples. The enhancer atlas is used to compare regulatory programs between different cell types and identify disease-associated regulatory SNPs, and will be a resource for studies on cell-type-specific enhancers. In the second paper, single-molecule sequencing is used to map human and mouse transcription start sites and their usage in a panel of distinct human and mouse primary cells, cell lines and tissues to produce the most comprehensive mammalian gene expression atlas to date. The data provide a plethora of insights into open reading frames and promoters across different cell types in addition to valuable annotation of mammalian cell-type-specific transcriptomes.

Knowledge of the complete genomic DNA sequence of an organism allows a systematic approach to defining its genetic components. The genomic sequence provides access to the complete structures of all genes, including those without known function, their control elements, and, by inference, the proteins they encode, as well as all other biologically important sequences. Furthermore, the sequence is a rich and permanent source of information for the design of further biological studies of the organism and for the study of evolution through cross-species sequence comparison. The power of this approach has been amply demonstrated by the determination of the sequences of a number of microbial and model organisms. The next step is to obtain the complete sequence of the entire human genome. Here we report the sequence of the euchromatic part of human chromosome 22. The sequence obtained consists of 12 contiguous segments spanning 33.4 megabases, contains at least 545 genes and 134 pseudogenes, and provides the first view of the complex chromosomal landscapes that will be found in the rest of the genome.

OBJECTIVE: To determine the frequency and pattern of lymph node metastasis (LNM) from papillary thyroid microcarcinoma (PTMC) and the results of node dissection, and to establish the optimal strategy for neck dissection in these patients. SUMMARY BACKGROUND DATA: Most PTMCs carry a favorable prognosis, but a few present with palpable lymphadenopathy. Patients with LNM are at risk for nodal recurrence, although they do not have higher mortality. The frequency and pattern of LNM from PTMC and the results of node dissection are not well established. METHODS: The frequency and pattern of LNM from 259 PTMCs were analyzed according to the size and location of the primary tumor. Of the 259, 24 with palpable nodes underwent therapeutic node dissection and the other 235 patients without palpable nodes underwent prophylactic node dissection. The authors compared the results of node dissection between the therapeutic group and the prophylactic group, and between PTMCs 5 mm or smaller and PTMCs larger than 5 mm. The authors also compared nodal recurrence between the prophylactic group and a no-lymph-node-dissection group (155 PTMCs). RESULTS: Overall, 64.1% (166/259) and 44.5% (93/209) had node involvement of the central and ipsilateral lateral compartment, respectively. Pretracheal (43.2%), ipsilateral central (36.3%), and ipsilateral mid-lower (37.8%) jugular were more commonly involved. LNM was more frequent in the therapeutic group than in the prophylactic group (95.8% vs. 60.9% for central compartment, 83.3% vs. 39.5% for ipsilateral lateral compartment). Nodal recurrence was more common in the therapeutic group than in the prophylactic group (16.7% vs. 0.43%), but did not differ between the prophylactic group and the no-dissection group (0.43% vs. 0.65%). The tumor size did not influence nodal recurrence. Nodal recurrence preferentially occurred in ipsilateral mid-lower jugular nodes. CONCLUSIONS: Patients who have PTMC presenting with palpable lymphadenopathy should have therapeutic node dissection. Prophylactic node dissection is not beneficial in those without palpable lymphadenopathy.

Exposure of platelets to shear stress leads to aggregation in the absence of exogenous agonists. We have now found that different adhesive proteins and platelet membrane glycoproteins are involved in aggregation depending on the shear stress condi- tions and the concentration of divalent cations in the medium. When blood is collected with trisodium citrate as anticoagu- lant, which causes a decrease in the levels of external ionized calcium (ICa2+I), platelet aggregation can be induced under low shear force (12 dyn/cm2) and is mediated by fibrinogen binding to the glycoprotein Hib-Mila complex. Aggregates formed under these conditions are not stable, and when shear force is in- creased to 68 dyn/cm2, disaggregation results. By contrast, platelets from blood collected with hirudin as anticoagulant, wherein ICa2+10 is within normal plasma levels, do not undergo low shear-induced aggregation; however, after exposure to a shear force above 80 dyn/cm2, aggregation is observed but only when von Willebrand factor is present and can interact with both its platelet binding sites, glycoprotein Ib-IX and glycopro- tein Jib-Mia. Fibrinogen is not involved in high shear-induced aggregation which, in fact, occurs normally in patients with severe afibrinogenemia. Thus, von Willebrand factor in the ab- sence of exogenous agonists can mediate platelet aggregation in experimental conditions that may mimic the hemorheological situation of partially occluded arteries. This pathway of platelet aggregation involving only one adhesive ligand and two mem- brane adhesion receptors may play a relevant role in thrombo- genesis. (

Alzheimer's disease (AD) is the most common form of age-related dementia, characterized by progressive memory loss and cognitive disturbance. Mutations of presenilin 1 (PS1) and presenilin 2 (PS2) are causative factors for autosomal-dominant early-onset familial AD (FAD). Induced pluripotent stem cell (iPSC) technology can be used to model human disorders and provide novel opportunities to study cellular mechanisms and establish therapeutic strategies against various diseases, including neurodegenerative diseases. Here we generate iPSCs from fibroblasts of FAD patients with mutations in PS1 (A246E) and PS2 (N141I), and characterize the differentiation of these cells into neurons. We find that FAD-iPSC-derived differentiated neurons have increased amyloid β42 secretion, recapitulating the molecular pathogenesis of mutant presenilins. Furthermore, secretion of amyloid β42 from these neurons sharply responds to γ-secretase inhibitors and modulators, indicating the potential for identification and validation of candidate drugs. Our findings demonstrate that the FAD-iPSC-derived neuron is a valid model of AD and provides an innovative strategy for the study of age-related neurodegenerative diseases.

Endoscopic resection (ER) includes endoscopic mucosal resection (EMR), wherein the affected mucosal lesion is held or aspirated and resected with a snare, and endoscopic submucosal dissection (ESD), which refers to en bloc resection of an extensive lesion using an IT knife or hook knife Other endoscopic treatments available include photodynamic therapy (PDT), argon plasma coagulation (APC), and electromagnetic coagulation therapy.

Purpose of the guidelines The primary objective of these guidelines is to provide general clinicians with information that would guide them to make informed choices of the available diagnosis/treatment strategies for esophageal cancer (intended for malignant esophageal tumors of epithelial origin, not for any other non-epithelial malignant tumors of the esophagus or metastatic esophageal malignant tumors). Furthermore, these guidelines are also intended as an aid for healthcare professionals other than the physicians, patients, and patients’ family members, to obtain an understanding of the fundamental principles of the diagnosis and treatment of esophageal cancer. These guidelines are intended to allow physicians to undertake diagnosis and treatment of esophageal cancer by sharing the information contained in the guidelines and promote mutual understanding among the healthcare professionals, patients, and their family members.

We often evaluate the self and others from social comparisons. We feel envy when the target person has superior and self-relevant characteristics. Schadenfreude occurs when envied persons fall from grace. To elucidate the neurocognitive mechanisms of envy and schadenfreude, we conducted two functional magnetic resonance imaging studies. In study one, the participants read information concerning target persons characterized by levels of possession and self-relevance of comparison domains. When the target person's possession was superior and self-relevant, stronger envy and stronger anterior cingulate cortex (ACC) activation were induced. In study two, stronger schadenfreude and stronger striatum activation were induced when misfortunes happened to envied persons. ACC activation in study one predicted ventral striatum activation in study two. Our findings document mechanisms of painful emotion, envy, and a rewarding reaction, schadenfreude.

An original technique for performing endoscopic thyroidectomy using a breast approach to avoid an operative scar in the neck was developed. The subcutaneous space in the breast area and the subplatysmal space in the neck were bluntly dissected through a 15-mm incision between the nipples, and CO2 was insufflated at 6 mm Hg to create the operative space. Three trocars were inserted at the breast, and dissection of the thyroid and division of the thyroid vessels and parenchyma were performed endoscopically using an ultrasonically activated scalpel. Four hemithyroidectomies and one partial resection of the thyroid for five female patients with thyroid adenomas 5 to 7 cm in diameter were successfully performed using this procedure. There were no conversions to open surgery or complications. No scars were apparent in the neck, and all patients were fully satisfied with the cosmetic results. Endoscopic thyroidectomy using a breast approach and low-pressure subcutaneous CO2 insufflation is a feasible and safe procedure, which results in satisfactory cosmetic results.

Mammalian lungs begin as an outpocket of the foregut, and depend on multiple stages of branching morphogenesis and alveogenesis to reach their final form. An examination of fgf receptor gene expression indicated that all four receptors (fgfr-1 to fgfr-4) are expressed in postnatal lungs at varying levels. We show that mice homozygous for a targeted mutation of fgfr-4 exhibited no overt abnormalities in the lungs or any other organ. However, mice doubly homozygous for disruptions of the fgfr-3 and fgfr-4 genes display novel phenotypes not present in either single mutant, which include pronounced dwarfism and lung abnormalities. Lungs of fgfr-3(-/-)fgfr-4(-/- )animals, which are normal at birth, are completely blocked in alveogenesis and do not form secondary septae to delimit alveoli. Consequently, air spaces in the lung are expanded and no alveoli can be seen. The mutant lungs failed to downregulate postnatal elastin deposition despite their normal levels of surfactant expression and cell proliferation. These data revealed a cooperative function of FGFR-3 and FGFR-4 to promote the formation of alveoli during postnatal lung development.

Adiponectin, which is secreted specifically by adipose tissue, has been shown to act as an anti-atherosclerotic protein by direct effects on endothelial cells. Clinical studies have shown that adiponectin levels are lower in individuals with obesity, diabetes and coronary artery disease. The present study investigated relationships between serum adiponectin levels and body mass index (BMI), blood pressure, insulin resistance index, lipid profile, uric acid and high-sensitivity C-reactive protein levels in a large number of Japanese subjects not taking any medication for metabolic disease and without severe illness (705 men and 262 women; age 30-65 years; BMI 22.5+/-2.9 kg/m(2)). The serum adiponectin concentration was measured by ELISA, without a protein-denaturing step. The insulin resistance index was assessed by homoeostasis model assessment (HOMA-IR). The serum concentration of adiponectin in women (13.5+/-7.9 microg/ml) was significantly higher than that in men (7.2+/-4.6 microg/ml). The serum adiponectin level was negatively correlated with BMI, systolic blood pressure, diastolic blood pressure, fasting plasma glucose, insulin, HOMA-IR, total cholesterol, triacylglycerols, low-density lipoprotein (LDL)-cholesterol and uric acid, and positively correlated with high-density lipoprotein (HDL)-cholesterol. The correlations between serum adiponectin level and insulin, HOMA-IR, triacylglycerols, HDL-cholesterol, LDL-cholesterol and uric acid were significant even after adjustment for age, sex and BMI. Stepwise multiple regression analysis revealed that HDL-cholesterol, sex, BMI and HOMA-IR were independently correlated with the serum adiponectin level (R(2)=0.377). These findings suggest that the serum adiponectin level is negatively correlated with HOMA-IR and positively correlated with HDL-cholesterol, independent of age, sex and BMI, in the Japanese population.

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are autoimmune skin diseases caused by autoantibodies against desmoglein (Dsg) 3 and Dsg1, respectively. Routine immunofluorescence testing of skin and serum from patients cannot distinguish between these two severe diseases since both have IgG Abs directed against keratinocyte cell surfaces. In this study, recombinant Dsg3 and Dsg1, produced as secreted proteins by baculovirus expression, have been utilized to develop ELISAs for the specific characterization of their autoantibodies. Of 49 PV sera, 46 were positive in the Dsg3 ELISA and 44 of 46 PF sera were positive in the Dsg1 ELISA, compared with only 3 of 23 sera of bullous pemphigoid, and none of 53 normal control sera in both ELISAs. Both the Dsg3 and Dsg1 ELISAs were more specific and sensitive than conventional immunofluorescence staining. These Ag-specific ELISAs revealed that more than one-half of PV sera (26 of 49) had anti-Dsg1 Abs in addition to anti-Dsg3 Abs. PV patients who had not only oral mucous lesions but also significant skin involvement tended to have higher titers of anti-Dsg1 Abs. Furthermore, the ELISA reactivity correlated well with clinical disease activity in 5 of 6 PV and 5 of 5 PF patients. This ELISA provides a sensitive and highly specific assay for the diagnosis of patients with PV and PF, the correlation of disease activity with serum Ab levels, and a novel tool for investigating the immunopathogenesis of pemphigus.

PURPOSE: To compare image quality obtained in phantoms with virtual monochromatic spectral (VMS) imaging with that obtained with conventional 120-kVp computed tomography (CT) for a given radiation dose. MATERIALS AND METHODS: Three syringes were filled with a diluted contrast medium (each syringe contained a contrast medium with a different iodine concentration [5, 10, or 15 mg of iodine per milliliter]), and a fourth syringe was filled with water. These syringes were placed in a torso phantom meant to simulate the standard human physique. The phantom was examined with a CT system and use of the fast kilovoltage switching (80 and 140 kVp) and conventional (120 kVp) modes. Image noise and contrast-to-noise (CNR) ratio were analyzed on VMS images and 120-kVp CT images. RESULTS: Image noise on VMS images in the range of 67-72 keV was significantly lower than that on the 120-kVp CT images (P < .014). Image noise was lowest at 69 keV and was 12% lower when compared with that on 120-kVp CT images. CNR on the VMS images was highest at 68 keV. CNR on the VMS images obtained at 68 keV in the syringes filled with diluted contrast material (5, 10, and 15 mg of iodine per milliliter) was 28%, 31%, and 30% higher, respectively, compared with that on the 120-kVp CT images (P < .001). CONCLUSION: VMS imaging at approximately 70 keV yielded lower image noise and higher CNR than did 120-kVp CT for a given radiation dose. VMS imaging has the potential to replace 120-kVp CT as the standard CT imaging modality, since optimal VMS imaging may be expected to yield improved image quality in a patient with standard body habitus.

Bipolar, amacrine, and ganglion cells of carp retina, stained intracellularly with Procion yellow, can be divided into types a and b, according to the destination of terminals and dendritic trees in the inner plexiform layer (sublamina a and b, respectively). Type a cells showed hyperpolarizing, or off, responses and type b cells depolarizing, or on, responses. Carp thus resembles cat in the basic organization of on and off pathways in the retina.

BACKGROUND AND AIMS: Immunosuppressive therapy with intravenous ciclosporin is an alternative treatment option to total colectomy for patients with ulcerative colitis (UC), while the benefits of oral administration of tacrolimus are not well defined and are based on reports of several uncontrolled studies. METHODS: Patients with refractory active UC were randomly assigned to a high trough concentration (10-15 ng/ml) group (HT group) (n = 21), low trough concentration (5-10 ng/ml) group (LT group) (n = 22), or placebo group (n = 20). Patients received an initial oral dose of 0.025 [DOSAGE ERROR CORRECTED] mg/kg tacrolimus or placebo twice daily. Efficacy was evaluated in 60 patients based on a disease activity index (DAI) score. Fifty eight patients had additional treatment with tacrolimus and were evaluated for efficacy in a 10 week open label extension. RESULTS: An improvement in DAI score (>or=4 points, all categories improved) was observed for 68.4% of cases in the HT group compared with 10.0% in the placebo group (p<0.001). In the HT group, 20.0% of patients had clinical remission and 78.9% had mucosal healing. In the open label extension, 55.2% of all patients had an improved DAI score at week 10. Mean dose of prednisolone was reduced from 19.7 mg/day at study entry to 7.8 mg/day at week 10. The incidence of side effects in the HT group was significantly higher than that of the placebo group (p = 0.043). The most common event was mild finger tremor. CONCLUSIONS: Our findings demonstrate dose dependent efficacy and safety of oral tacrolimus for remission-induction therapy of refractory UC. The optimal target range appears to be 10-15 ng/ml in terms of efficacy with two week therapy.

Metformin is an oral hypoglycemic agent which is most widely used as first-line therapy for type 2 diabetes. Metformin improves hyperglycemia by suppressing hepatic glucose production and increasing glucose uptake in muscle. Metformin also has been shown to reduce cardiovascular events in randomized controlled trials; however, the underlying mechanism remains to be established. Recent preclinical and clinical studies have suggested that metformin not only improves chronic inflammation through the improvement of metabolic parameters such as hyperglycemia, insulin resistance and atherogenic dyslipidemia, but also has a direct anti-inflammatory action. Studies have suggested that metformin suppresses inflammatory response by inhibition of nuclear factor κB (NFκB) via AMP-activated protein kinase (AMPK)-dependent and independent pathways. This review summarizes the basic and clinical evidence of the anti-inflammatory action of metformin and discusses its clinical implication.

BACKGROUND: While tumour necrosis factor alpha (TNF-alpha) appears to be associated with the development of non-alcoholic steatohepatitis (NASH), its precise role in the pathogenesis of NASH is not well understood. METHODS: Male mice deficient in both TNF receptors 1 (TNFR1) and 2 (TNFR2) (TNFRDKO mice) and wild-type mice were fed a methionine and choline deficient (MCD) diet or a control diet for eight weeks, maintaining isoenergetic intake. RESULTS: MCD dietary feeding of TNFRDKO mice for eight weeks resulted in attenuated liver steatosis and fibrosis compared with control wild-type mice. In the liver, the number of activated hepatic Kupffer cells recruited was significantly decreased in TNFRDKO mice after MCD dietary feeding. In addition, hepatic induction of TNF-alpha, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 was significantly suppressed in TNFRDKO mice. While in control animals MCD dietary feeding dramatically increased mRNA expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) in both whole liver and hepatic stellate cells, concomitant with enhanced activation of hepatic stellate cells, both factors were significantly lower in TNFRDKO mice. In primary cultures, TNF-alpha administration enhanced TIMP-1 mRNA expression in activated hepatic stellate cells and suppressed apoptotic induction in activated hepatic stellate cells. Inhibition of TNF induced TIMP-1 upregulation by TIMP-1 specific siRNA reversed the apoptotic suppression seen in hepatic stellate cells. CONCLUSIONS: Enhancement of the TNF-alpha/TNFR mediated signalling pathway via activation of Kupffer cells in an autocrine or paracrine manner may be critically involved in the pathogenesis of liver fibrosis in this NASH animal model.

Selective destruction of senescent cells Senescent cells are associated with a variety of age-related medical conditions and thus have been proposed as potential targets for therapy, but we do not yet have a full understanding of the underlying mechanisms. Johmura et al. used RNA interference to screen for enzymes essential to the survival of senescent cells (see the Perspective by Pan and Locasale). The authors identified a key role for glutamine metabolism, particularly the enzyme glutaminase 1, and demonstrated that inhibition of this pathway induced the death of senescent cells. Glutaminase targeting also ameliorated aging-related organ dysfunction and obesity-related disorders in mouse models, suggesting the potential therapeutic value of this approach. Science , this issue p. 265 ; see also p. 234

Oxidative stress has long been linked to the pathogenesis of neurodegenerative diseases; however, whether it is a cause or merely a consequence of the degenerative process is still unknown. We show that mice deficient in Cu, Zn-superoxide dismutase (SOD1) have features typical of age-related macular degeneration in humans. Investigations of senescent Sod1(-/-) mice of different ages showed that the older animals had drusen, thickened Bruch's membrane, and choroidal neovascularization. The number of drusen increased with age, and exposure of young Sod1(-/-) mice to excess light induced drusen. The retinal pigment epithelial cells of Sod1(-/-) mice showed oxidative damage, and their beta-catenin-mediated cellular integrity was disrupted, suggesting that oxidative stress may affect the junctional proteins necessary for the barrier integrity of the retinal pigment epithelium. These observations strongly suggest that oxidative stress may play a causative role in age-related retinal degeneration, and our findings provide evidence for the free radical theory of aging. In addition, these results demonstrate that the Sod1(-/-) mouse is a valuable animal model to study human age-related macular degeneration.