Tokyo University of Pharmacy and Life Sciences

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

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.

The tumor suppressor p53 is degraded by the ubiquitin‐proteasome system. p53 was polyubiquitinated in the presence of E1, UbcH5 as E2 and MDM2 oncoprotein. A ubiquitin molecule bound MDM2 through sulfhydroxy bond which is characteristic of ubiquitin ligase (E3)‐ubiquitin binding. The cysteine residue in the carboxyl terminus of MDM2 was essential for the activity. These data suggest that the MDM2 protein, which is induced by p53, functions as a ubiquitin ligase, E3, in human papillomavirus‐uninfected cells which do not have E6 protein.

We describe the draft genome of the microcrustacean Daphnia pulex, which is only 200 megabases and contains at least 30,907 genes. The high gene count is a consequence of an elevated rate of gene duplication resulting in tandem gene clusters. More than a third of Daphnia's genes have no detectable homologs in any other available proteome, and the most amplified gene families are specific to the Daphnia lineage. The coexpansion of gene families interacting within metabolic pathways suggests that the maintenance of duplicated genes is not random, and the analysis of gene expression under different environmental conditions reveals that numerous paralogs acquire divergent expression patterns soon after duplication. Daphnia-specific genes, including many additional loci within sequenced regions that are otherwise devoid of annotations, are the most responsive genes to ecological challenges.

Severe acute respiratory syndrome (SARS) is caused by a newly emerged coronavirus that infected more than 8000 individuals and resulted in more than 800 (10-15%) fatalities in 2003. The causative agent of SARS has been identified as a novel human coronavirus (SARS-CoV), and its viral protease, SARS-CoV 3CL(pro), has been shown to be essential for replication and has hence been recognized as a potent drug target for SARS infection. Currently, there is no effective treatment for this epidemic despite the intensive research that has been undertaken since 2003 (over 3500 publications). This perspective focuses on the status of various efficacious anti-SARS-CoV 3CL(pro) chemotherapies discovered during the last 12 years (2003-2015) from all sources, including laboratory synthetic methods, natural products, and virtual screening. We describe here mainly peptidomimetic and small molecule inhibitors of SARS-CoV 3CL(pro). Attempts have been made to provide a complete description of the structural features and binding modes of these inhibitors under many conditions.

The effects of uniform steady state (DC) extracellular electric fields on neuronal excitability were characterized in rat hippocampal slices using field, intracellular and voltage-sensitive dye recordings. Small electric fields (</40/ mV mm(-1)), applied parallel to the somato-dendritic axis, induced polarization of CA1 pyramidal cells; the relationship between applied field and induced polarization was linear (0.12 +/- 0.05 mV per mV mm(-1) average sensitivity at the soma). The peak amplitude and time constant (15-70 ms) of membrane polarization varied along the axis of neurons with the maximal polarization observed at the tips of basal and apical dendrites. The polarization was biphasic in the mid-apical dendrites; there was a time-dependent shift in the polarity reversal site. DC fields altered the thresholds of action potentials evoked by orthodromic stimulation, and shifted their initiation site along the apical dendrites. Large electric fields could trigger neuronal firing and epileptiform activity, and induce long-term (>1 s) changes in neuronal excitability. Electric fields perpendicular to the apical-dendritic axis did not induce somatic polarization, but did modulate orthodromic responses, indicating an effect on afferents. These results demonstrate that DC fields can modulate neuronal excitability in a time-dependent manner, with no clear threshold, as a result of interactions between neuronal compartments, the non-linear properties of the cell membrane, and effects on afferents.

This is the first publication on the distribution and sources of polycyclic aromatic hydrocarbons (PAHs) in riverine and coastal sediments in South East Asia where the rapid transfer of land-based pollutants into aquatic environments by heavy rainfall and runoff waters is of great concern. Twenty-nine Malaysian riverine and coastal sediments were analyzed for PAHs (3-7 rings) by gas chromatography mass spectrometry. Total PAHs concentrations in the sediment ranged from 4 to 924 ng/g. Alkylated homologues were abundant for all sediment samples. The ratio of the sum of methylphenanthrenes to phenanthrene (MP/P), an index of petrogenic PAHs contribution, was more than unity for 26 sediment samples and more than 3 for seven samples for urban rivers covering a broad range of locations. The MP/P ratio showed a strong correlation with the total PAHs concentrations, with an r2 value of 0.74. This ratio and all other compositional features indicated that Malaysian urban sediments are heavily impacted by petrogenic PAHs. This finding is in contrast to other studies reported in many industrialized countries where PAHs are mostly of pyrogenic origin. The MP/P ratio was also significantly correlated with higher molecular weight PAHs such as benzo[a]pyrene, suggesting unique PAHs source in Malaysia which contains both petrogenic PAHs and pyrogenic PAHs. PAHs and hopanes fingerprints indicated that used crankcase oil is one of the major contributors of the sedimentary PAHs. Two major routes of inputs to aquatic environments have been identified: (1) spillage and dumping of waste crankcase oil and (2) leakage of crankcase oils from vehicles onto road surfaces, with the subsequent washout by street runoff. N-Cyclohexyl-2-benzothiazolamine (NCBA), a molecular marker of street dust, was detected in the polluted sediments. NCBA and other biomarker profiles confirmed our hypothesis of the input from street dust contained the leaked crankcase oil. The fingerprints excluded crude oil, fresh lubricating oil, asphalt, and tire-particles as major contributors.

ABCG2, also known as BCRP, is a high-capacity urate exporter, the dysfunction of which raises gout/hyperuricemia risk. Generally, hyperuricemia has been classified into urate 'overproduction type' and/or 'underexcretion type' based solely on renal urate excretion, without considering an extra-renal pathway. Here we show that decreased extra-renal urate excretion caused by ABCG2 dysfunction is a common mechanism of hyperuricemia. Clinical parameters, including urinary urate excretion, are examined in 644 male outpatients with hyperuricemia. Paradoxically, ABCG2 export dysfunction significantly increases urinary urate excretion and risk ratio of urate overproduction. Abcg2-knockout mice show increased serum uric acid levels and renal urate excretion, and decreased intestinal urate excretion. Together with high ABCG2 expression in extra-renal tissues, our data suggest that the 'overproduction type' in the current concept of hyperuricemia be renamed 'renal overload type', which consists of two subtypes—'extra-renal urate underexcretion' and genuine 'urate overproduction'—providing a new concept valuable for the treatment of hyperuricemia and gout. Hyperuricemia, or gout, is thought to arise either from urate overproduction or from decreased renal excretion of urate. Ichidaet al. show that the extra-renal excretion of urate also has a role in the pathogenesis of hyperuricemia, and propose a new classification for patients with this disease.

In anaerobic biota, reducing equivalents (electrons) are transferred between different species of microbes [interspecies electron transfer (IET)], establishing the basis of cooperative behaviors and community functions. IET mechanisms described so far are based on diffusion of redox chemical species and/or direct contact in cell aggregates. Here, we show another possibility that IET also occurs via electric currents through natural conductive minerals. Our investigation revealed that electrically conductive magnetite nanoparticles facilitated IET from Geobacter sulfurreducens to Thiobacillus denitrificans, accomplishing acetate oxidation coupled to nitrate reduction. This two-species cooperative catabolism also occurred, albeit one order of magnitude slower, in the presence of Fe ions that worked as diffusive redox species. Semiconductive and insulating iron-oxide nanoparticles did not accelerate the cooperative catabolism. Our results suggest that microbes use conductive mineral particles as conduits of electrons, resulting in efficient IET and cooperative catabolism. Furthermore, such natural mineral conduits are considered to provide ecological advantages for users, because their investments in IET can be reduced. Given that conductive minerals are ubiquitously and abundantly present in nature, electric interactions between microbes and conductive minerals may contribute greatly to the coupling of biogeochemical reactions.

Methanogenesis is an essential part of the global carbon cycle and a key bioprocess for sustainable energy. Methanogenesis from organic matter is accomplished by syntrophic interactions among different species of microbes, in which interspecies electron transfer (IET) via diffusive carriers (e.g. hydrogen and formate) is known to be the bottleneck step. We report herein that the supplementation of soil microbes with (semi)conductive iron-oxide minerals creates unique interspecies interactions and facilitates methanogenesis. Methanogenic microbes were enriched from rice paddy field soil with either acetate or ethanol as a substrate in the absence or presence of (semi)conductive iron oxides (haematite or magnetite). We found that the supplementation with either of these iron oxides resulted in the acceleration of methanogenesis in terms of lag time and production rate, while the supplementation with an insulative iron oxide (ferrihydrite) did not. Clone-library analyses of 16S rRNA gene fragments PCR-amplified from the enrichment cultures revealed that the iron-oxide supplementation stimulated the growth of Geobacter spp. Furthermore, the addition of a specific inhibitor for methanogenesis suppressed the growth of Geobacter spp. These results suggest that Geobacter grew under syntrophic association with methanogens, and IET could occur via electric currents through (semi)conductive iron-oxide minerals (termed 'electric syntrophy'). Given the ubiquity of conductive minerals in nature, such energetic interactions may occur widely in soil and sediments and can be used to develop efficient bioenergy processes.

Exosomes are small endosome-derived vesicles containing a wide range of functional proteins, mRNA, and miRNA. Exosomal miRNA from cancer cells helps modulate the microenvironment. In multiple myeloma (MM), the massive proliferation of malignant plasma cells causes hypoxia. To date, the majority of in vitro hypoxia studies of cancer cells have used acute hypoxic exposure (3-24 hours). Thus, we attempted to clarify the role of MM-derived exosomes in hypoxic bone marrow by using MM cells grown continuously in vitro under chronic hypoxia (hypoxia-resistant MM [HR-MM] cells). The HR-MM cells produced more exosomes than the parental cells under normoxia or acute hypoxia conditions, and miR-135b was significantly upregulated in exosomes from HR-MM cells. Exosomal miR-135b directly suppressed its target factor-inhibiting hypoxia-inducible factor 1 (FIH-1) in endothelial cells. Finally, exosomal miR-135b from HR-MM cells enhanced endothelial tube formation under hypoxia via the HIF-FIH signaling pathway. This in vitro HR myeloma cell model will be useful for investigating MM cell-endothelial cell interactions under hypoxic conditions, which may mimic the in vivo bone marrow microenvironment. Although tumor angiogenesis is regulated by various factors, exosomal miR-135b may be a target for controlling MM angiogenesis.

Phosphoinositide-specific phospholipase C is an effector molecule in the signal transduction process. It generates two second messengers, inositol-1,4,5-trisphosphate and diacylglycerol from phosphatidylinositol 4,5-bisphosphate. Currently, thirteen mammal PLC isozymes have been identified, and they are divided into six groups: PLC-beta, -gamma, -delta, -epsilon, -zeta and -eta. Sequence analysis studies demonstrated that each isozyme has more than one alternative splicing variant. PLC isozymes contain the X and Y domains that are responsible for catalytic activity. Several other domains including the PH domain, the C2 domain and EF hand motifs are involved in various biological functions of PLC isozymes as signaling proteins. The distribution of PLC isozymes is tissue and organ specific. Recent studies on isolated cells and knockout mice depleted of PLC isozymes have revealed their distinct phenotypes. Given the specificity in distribution and cellular localization, it is clear that each PLC isozyme bears a unique function in the modulation of physiological responses. In this review, we discuss the structural organization, enzymatic properties and molecular diversity of PLC splicing variants and study functional and physiological roles of each isozyme.

We have established a high-efficiency method for transforming the unicellular, green alga Chlamydomonas reinhardtii by electroporation. Electroporation of strains CC3395 and CC425, cell wall-less mutants devoid of argininosuccinate lyase (encoded by ARG7), in the presence of the plasmid pJD67 (which contains ARG7) was used to optimize conditions for the introduction of exogenous DNA. The conditions that were varied included osmolarity, temperature, concentration of exogenous DNA, voltage and capacitance. Following optimization, the maximum transformation frequency obtained was 2 x 10(5) transformants per microg of DNA; this frequency is two orders of magnitude higher than obtained with the current standard method using glass beads to introduce exogenous DNA. The electroporation procedure described in this article is of general utility, and makes it feasible to isolate genes by direct complementation of Chlamydomonas reinhardtii mutants.

In situ hydrogels have attracted considerable attention in tissue engineering because of their minimal invasiveness and ability to match the irregular tissue defects. However, hydrous physiological environments and the high level of moisture in hydrogels severely hamper binding to the target tissue and easily cause wound infection, thereby limiting the effectiveness in wound care management. Thus, forming an intimate assembly of the hydrogel to the tissue and preventing wound infecting still remains a significant challenge. In this study, inspired by mussel adhesive protein, a biomimetic dopamine‐modified ε‐poly‐ l ‐lysine‐polyethylene glycol‐based hydrogel (PPD hydrogel) wound dressing is developed in situ using horseradish peroxidase cross‐linking. The biomimetic catechol–Lys residue distribution in PPD polymer provides a catechol–Lys cooperation effect, which endows the PPD hydrogels with superior wet tissue adhesion properties. It is demonstrated that the PPD hydrogel can facilely and intimately integrate with biological tissue and exhibits superior capacity of in vivo hemostatic and accelerated wound repair. In addition, the hydrogels exhibit outstanding anti‐infection property because of the inherent antibacterial ability of ε‐poly‐ l ‐lysine. These findings shed new light on the development of mussel‐inspired tissue‐anchored and antibacterial hydrogel materials serving as wound dressings.

Gout based on hyperuricemia is a common disease with a genetic predisposition, which causes acute arthritis. The ABCG2/BCRP gene, located in a gout-susceptibility locus on chromosome 4q, has been identified by recent genome-wide association studies of serum uric acid concentrations and gout. Urate transport assays demonstrated that ABCG2 is a high-capacity urate secretion transporter. Sequencing of the ABCG2 gene in 90 hyperuricemia patients revealed several nonfunctional ABCG2 mutations, including Q126X. Quantitative trait locus analysis of 739 individuals showed that a common dysfunctional variant of ABCG2, Q141K, increases serum uric acid. Q126X is assigned to the different disease haplotype from Q141K and increases gout risk, conferring an odds ratio of 5.97. Furthermore, 10% of gout patients (16 out of 159 cases) had genotype combinations resulting in more than 75% reduction of ABCG2 function (odds ratio, 25.8). Our findings indicate that nonfunctional variants of ABCG2 essentially block gut and renal urate excretion and cause gout.

Tardigrades, also known as water bears, are small aquatic animals. Some tardigrade species tolerate almost complete dehydration and exhibit extraordinary tolerance to various physical extremes in the dehydrated state. Here we determine a high-quality genome sequence of Ramazzottius varieornatus, one of the most stress-tolerant tardigrade species. Precise gene repertoire analyses reveal the presence of a small proportion (1.2% or less) of putative foreign genes, loss of gene pathways that promote stress damage, expansion of gene families related to ameliorating damage, and evolution and high expression of novel tardigrade-unique proteins. Minor changes in the gene expression profiles during dehydration and rehydration suggest constitutive expression of tolerance-related genes. Using human cultured cells, we demonstrate that a tardigrade-unique DNA-associating protein suppresses X-ray-induced DNA damage by ∼40% and improves radiotolerance. These findings indicate the relevance of tardigrade-unique proteins to tolerability and tardigrades could be a bountiful source of new protection genes and mechanisms.

Olfactory receptors (ORs) detect odors in the environment, and OR genes constitute the largest multigene family in mammals. Numbers of OR genes vary greatly among species—reflecting the respective species' lifestyles—and this variation is caused by frequent gene gains and losses during evolution. However, whether the extent of gene gains/losses varies among individual gene lineages and what might generate such variation is unknown. To answer these questions, we used a newly developed phylogeny-based method to classify &gt;10,000 intact OR genes from 13 placental mammal species into 781 orthologous gene groups (OGGs); we then compared the OGGs. Interestingly, African elephants had a surprisingly large repertoire (∼2000) of functional OR genes encoded in enlarged gene clusters. Additionally, OR gene lineages that experienced more gene duplication had weaker purifying selection, and Class II OR genes have evolved more dynamically than those in Class I. Some OGGs were highly expanded in a lineage-specific manner, while only three OGGs showed complete one-to-one orthology among the 13 species without any gene gains/losses. These three OGGs also exhibited highly conserved amino acid sequences; therefore, ORs in these OGGs may have physiologically important functions common to every placental mammal. This study provides a basis for inferring OR functions from evolutionary trajectory.

We report the design, synthesis, and evaluation of a new type of non-precious-metal catalyst made from network polymers. 2,6-Diaminopyridine was selected as a building-block monomer for the formation of a nitrogen-rich network polymer that forms self-supporting spherical backbone structures and contains a high density of metal-coordination sites. A Co-/Fe-coordinating pyrolyzed polymer exhibited a high specific oxygen reduction activity with onset and half-wave potentials of 0.87 and 0.76 V vs RHE, respectively, in neutral media. There was no crossover effect of organics on its activity. The power output of a microbial fuel cell equipped with this catalyst on its cathode was more than double the output with a commercial 20 wt % Pt/C catalyst.

PGE2 functions as a potent stimulator of bone resorption. The action of PGE2 is thought to be mediated by some PGE receptor subtypes present in osteoblastic cells. In this study, we examined the involvement of PGE receptor subtypes, EP1, EP2, EP3, and EP4, in PGE2-induced bone resorption using specific agonists for the respective EPs. In mouse calvaria cultures, EP4 agonist markedly stimulated bone resorption, but its maximal stimulation was less than that induced by PGE2. EP2 agonist also stimulated bone resorption, but only slightly. EP1 and EP3 agonists did not stimulate it at all. RT-PCR showed that osteoblastic cells isolated from newborn mouse calvaria expressed all of the EPs messenger RNA (mRNA). Both EP2 agonist and EP4 agonist induced cAMP production and the expression of osteoclast differentiation factor (ODF) mRNA in osteoblastic cells. Simultaneous addition of EP2 and EP4 agonists cooperatively induced cAMP production and ODF mRNA expression. In mouse bone marrow cultures, EP2 and EP4 agonists moderately induced osteoclast formation, but the simultaneous addition of the two agonists cooperatively induced it, similar to that by PGE2. In calvaria culture from EP4 knockout mice, a marked reduction in bone resorption to PGE2 was found. In EP4 knockout mice, EP4 agonist failed to induce bone resorption, but EP2 agonist slightly, but significantly, induced bone resorption. These findings suggest that PGE2 stimulates bone resorption by a mechanism involving cAMP and ODF, which is mediated mainly by EP4 and partially by EP2.

Toll-like receptors (TLRs) are innate recognition molecules for microbial products, but their direct interactions with corresponding ligands remain unclarified. LPS, a membrane constituent of gram-negative bacteria, is the best-studied TLR ligand and is recognized by TLR4 and MD-2, a molecule associated with the extracellular domain of TLR4. Although TLR4-MD-2 recognizes LPS, little is known about the physical interaction between LPS and TLR4-MD-2. Here, we demonstrate cell surface LPS-TLR4-MD-2 complexes. CD14 greatly enhances the formation of LPS-TLR4-MD-2 complexes, but is not coprecipitated with LPS-TLR4-MD-2 complexes, suggesting a role for CD14 in LPS loading onto TLR4-MD-2 but not in the interaction itself between LPS and TLR4-MD-2. A tentative dissociation constant (Kd) for LPS-TLR4-MD-2 complexes was approximately 3 nM, which is approximately 10-20 times lower than the reported Kd for LPS-MD-2 or LPS-CD14. The presence of detergent disrupts LPS interaction with CD14 but not with TLR4-MD-2. E5531, a lipid A antagonist developed for therapeutic intervention of endotoxin shock, blocks LPS interaction with TLR4-MD-2 at a concentration 100 times lower than that required for blocking LPS interaction with CD14. These results reveal direct LPS interaction with cell surface TLR4-MD-2 that is distinct from that with MD-2 or CD14.