Thalidomide and its metabolite 5-hydroxythalidomide induce teratogenicity via the cereblon neosubstrate PLZF.

Thalidomide causes teratogenic effects by inducing protein degradation via cereblon (CRBN)-containing ubiquitin ligase and modification of its substrate specificity. Human P450 cytochromes convert thalidomide into two monohydroxylated metabolites that are considered to contribute to thalidomide effects, through mechanisms that remain unclear. Here, we report that promyelocytic leukaemia zinc finger (PLZF)/ZBTB16 is a CRBN target protein whose degradation is involved in thalidomide- and 5-hydroxythalidomide-induced teratogenicity. Using a human transcription factor protein array produced in a wheat cell-free protein synthesis system, PLZF was identified as a thalidomide-dependent CRBN substrate. PLZF is degraded by the ubiquitin ligase CRL4CRBN in complex with thalidomide, its derivatives or 5-hydroxythalidomide in a manner dependent on the conserved first and third zinc finger domains of PLZF. Surprisingly, thalidomide and 5-hydroxythalidomide confer distinctly different substrate specificities to mouse and chicken CRBN, and both compounds cause teratogenic phenotypes in chicken embryos. Consistently, knockdown of Plzf induces short bone formation in chicken limbs. Most importantly, degradation of PLZF protein, but not of the known thalidomide-dependent CRBN substrate SALL4, was induced by thalidomide or 5-hydroxythalidomide treatment in chicken embryos. Furthermore, PLZF overexpression partially rescued the thalidomide-induced phenotypes. Our findings implicate PLZF as an important thalidomide-induced CRBN neosubstrate involved in thalidomide teratogenicity.

Role of hepcidin upregulation and proteolytic cleavage of ferroportin 1 in hepatitis C virus-induced iron accumulation.

Hepatitis C virus (HCV) is a pathogen characterized not only by its persistent infection leading to the development of cirrhosis and hepatocellular carcinoma (HCC), but also by metabolic disorders such as lipid and iron dysregulation. Elevated iron load is commonly observed in the livers of patients with chronic hepatitis C, and hepatic iron overload is a highly profibrogenic and carcinogenic factor that increases the risk of HCC. However, the underlying mechanisms of elevated iron accumulation in HCV-infected livers remain to be fully elucidated. Here, we observed iron accumulation in cells and liver tissues under HCV infection and in mice expressing viral proteins from recombinant adenoviruses. We established two molecular mechanisms that contribute to increased iron load in cells caused by HCV infection. One is the transcriptional induction of hepcidin, the key hormone for modulating iron homeostasis. The transcription factor cAMP-responsive element-binding protein hepatocyte specific (CREBH), which was activated by HCV infection, not only directly recognizes the hepcidin promoter but also induces bone morphogenetic protein 6 (BMP6) expression, resulting in an activated BMP-SMAD pathway that enhances hepcidin promoter activity. The other is post-translational regulation of the iron-exporting membrane protein ferroportin 1 (FPN1), which is cleaved between residues Cys284 and Ala285 in the intracytoplasmic loop region of the central portion mediated by HCV NS3-4A serine protease. We propose that host transcriptional activation triggered by endoplasmic reticulum stress and FPN1 cleavage by viral protease work in concert to impair iron efflux, leading to iron accumulation in HCV-infected cells.

Protein-protein interactions between jasmonate-related master regulator MYC and transcriptional mediator MED25 depend on a short binding domain.

A network of protein-protein interactions (PPI) is involved in the activation of (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile), a plant hormone that regulates plant defense responses as well as plant growth and development. In the absence of JA-Ile, inhibitory protein jasmonate-ZIM-domain (JAZ) represses JA-related transcription factors, including a master regulator, MYC. In contrast, when JA-Ile accumulates in response to environmental stresses, PPI occurs between JAZ and the F-box protein COI1, which triggers JAZ degradation, resulting in derepressed MYC that can interact with the transcriptional mediator MED25 and upregulate JA-Ile-related gene expression. Activated JA signaling is eventually suppressed through the catabolism of JA-Ile and feedback suppression by JAZ splice variants containing a cryptic MYC-interacting domain (CMID). However, the detailed structural basis of some PPIs involved in JA-Ile signaling remains unclear. Herein, we analyzed PPI between MYC3 and MED25, focusing on the key interactions that activate the JA-Ile signaling pathway. Biochemical assays revealed that a short binding domain of MED25 (CMIDM) is responsible for the interaction with MYC, and that a bipartite interaction is critical for the formation of a stable complex. We also show the mode of interaction between MED25 and MYC is closely related to that of CMID and MYC. In addition, quantitative analyses on the binding of MYC3-JAZs and MYC3-MED25 revealed the order of binding affinity as JAZJas < MED25CMIDM < JAZCMID, suggesting a mechanism for how the transcriptional machinery causes activation and negative feedback regulation during jasmonate signaling. These results further illuminate the transcriptional machinery responsible for JA-Ile signaling.

A simple method for labeling proteins and antibodies with biotin using the proximity biotinylation enzyme TurboID.

Proteins and antibodies labeled with biotin have been widely used for protein analysis, enzyme immunoassays, and diagnoses. Presently, they are prepared using either a chemical reaction involving a biotin N-hydroxysuccinimide (NHS) ester compound or by enzymatic biotin ligation using a combination of a biotinylation-peptide tag and Escherichia coli BirA. However, these methods are relatively complicated. Recently BirA was improved to TurboID, a highly active enzyme for proximity labeling with biotin. Here, we demonstrate a novel simple biotin labeling method for proteins and antibodies using TurboID. Purified TurboID was mixed with a protein or an antibody in the presence of biotin and ATP in the general biochemical buffer condition, followed by biotin labeling. Biotin labeling sites by TurboID were found on the surface of green fluorescent protein. Biotin labeling of IκBα by TurboID indicated its binding to RelA. Furthermore, TurboID-dependent biotin labeling of monoclonal antibodies from rabbits and mice could be directly used for immunoblotting detection of specific proteins without the purification step. These results indicate that TurboID provides a very useful and simple method for biotin labeling of functional proteins.

Inattentive Driving Detection Using Body-Worn Sensors: Feasibility Study.

This study aims to build a system for detecting a driver's internal state using body-worn sensors. Our system is intended to detect inattentive driving that occurs during long-term driving on a monotonous road, such as a high-way road. The inattentive state of a driver in this study is an absent-minded state caused by a decrease in driver vigilance levels due to fatigue or drowsiness. However, it is difficult to clearly define these inattentive states because it is difficult for the driver to recognize when they fall into an absent-minded state. To address this problem and achieve our goal, we have proposed a detection algorithm for inattentive driving that not only uses a heart rate sensor, but also uses body-worn inertial sensors, which have the potential to detect driver behavior more accurately and at a much lower cost. The proposed method combines three detection models: body movement, drowsiness, and inattention detection, based on an anomaly detection algorithm. Furthermore, we have verified the accuracy of the algorithm with the experimental data for five participants that were measured in long-term and monotonous driving scenarios by using a driving simulator. The results indicate that our approach can detect both the inattentive and drowsiness states of drivers using signals from both the heart rate sensor and accelerometers placed on wrists.

CIPK23 regulates blue light-dependent stomatal opening in Arabidopsis thaliana.

Phototropins (phot1 and phot2) are plant blue light receptor kinases that function to mediate phototropism, chloroplast movement, leaf flattening, and stomatal opening in Arabidopsis. Considerable progress has been made in understanding the mechanisms associated with phototropin receptor activation by light. However, the identities of phototropin signaling components are less well understood by comparison. In this study, we specifically searched for protein kinases that interact with phototropins by using an in vitro screening method (AlphaScreen) to profile interactions against an Arabidopsis protein kinase library. We found that CBL-interacting protein kinase 23 (CIPK23) interacts with both phot1 and phot2. Although these interactions were verified by in vitro pull-down and in vivo bimolecular fluorescence complementation assays, CIPK23 was not phosphorylated by phot1, as least in vitro. Mutants lacking CIPK23 were found to exhibit impaired stomatal opening in response to blue light but no deficits in other phototropin-mediated responses. We further found that blue light activation of inward-rectifying K+ (K+ in ) channels was impaired in the guard cells of cipk23 mutants, whereas activation of the plasma membrane H+ -ATPase was not. The blue light activation of K+ in channels was also impaired in the mutant of BLUS1, which is one of the phototropin substrates in guard cells. We therefore conclude that CIPK23 promotes stomatal opening through activation of K+ in channels most likely in concert with BLUS1, but through a mechanism other than activation of the H+ -ATPase. The role of CIPK23 as a newly identified component of phototropin signaling in stomatal guard cells is discussed.

The E3 ubiquitin ligase MIB2 enhances inflammation by degrading the deubiquitinating enzyme CYLD.

The tumor suppressor CYLD is a deubiquitinating enzyme that suppresses polyubiquitin-dependent signaling pathways, including the proinflammatory and cell growth-promoting NF-κB pathway. Missense mutations in the CYLD gene are present in individuals with syndromes such as multiple familial trichoepithelioma (MFT), but the pathogenic roles of these mutations remain unclear. Recent studies have shown that CYLD interacts with a RING finger domain protein, mind bomb homologue 2 (MIB2), in the regulation of NOTCH signaling. However, whether MIB2 is an E3 ubiquitin ligase that acts on CYLD is unknown. Here, using the cell-free-based AlphaScreen and pulldown assays to detect protein-protein interactions, along with immunofluorescence assays and murine Mib2 knockout cells and animals, we demonstrate that MIB2 promotes proteasomal degradation of CYLD and enhances NF-κB signaling. Of note, arthritic inflammation was suppressed in Mib2-deficient mice. We further observed that the ankyrin repeat in MIB2 interacts with the third CAP domain in CYLD and that MIB2 catalyzes Lys-48-linked polyubiquitination of CYLD at Lys-338 and Lys-530. MIB2-dependent CYLD degradation activated NF-κB signaling via tumor necrosis factor alpha (TNFα) stimulation and the linear ubiquitination assembly complex (LUBAC). Mib2-knockout mice had reduced serum interleukin-6 (IL-6) and exhibited suppressed inflammatory responses in the K/BxN serum-transfer arthritis model. Interestingly, MIB2 significantly enhanced the degradation of a CYLDP904L variant identified in an individual with MFT, although the molecular pathogenesis of the disease was not clarified here. Together, these results suggest that MIB2 enhances NF-κB signaling in inflammation by promoting the ubiquitin-dependent degradation of CYLD.

Subquinocin, a small molecule inhibitor of CYLD and USP-family deubiquitinating enzymes, promotes NF-κB signaling.

The tumor suppressor CYLD negatively regulates polyubiquitination-dependent cellular signaling such as nuclear factor (NF)-κB signaling. In addition to CYLD, multiple deubiquitinating enzymes (DUBs) are also involved in the regulation of this signaling pathway, and distinct role of CYLD is yet to be clarified. Here, we identified a small chemical named Subquinocin that inhibited the DUB activity of recombinant CYLD using a wheat cell-free protein synthesis and an AlphaScreen technology. In cells, Subquinocin increased the polyubiquitination of NEMO and RIP1 and enhanced NF-κB activation. Modeling and mutation analyses indicated that Subquinocin interacted with Y940 in CYLD, which locates close to catalytic center of CYLD, and is conserved among the USP-family DUBs. Further biochemical evaluation revealed that Subquinocin inhibited USP-family DUBs, but not other family DUBs including OTU. Although Subquinocin showed a broad specificity toward USP-family DUBs, the inhibitory effect of Subquinocin on NF-κB signaling was negligible in CYLD-KO cells, indicating that CYLD is a major target of Subquinocin on the suppression of NF-κB signaling. In conclusion, Subquinocin identified here is a useful tool to analyze the signal transduction mediated by USP-family DUBs.

Raf-like kinases CBC1 and CBC2 negatively regulate stomatal opening by negatively regulating plasma membrane H+-ATPase phosphorylation in Arabidopsis.

Stomatal pores, which are surrounded by pairs of guard cells in the plant epidermis, regulate gas exchange between plants and the atmosphere, thereby controlling photosynthesis and transpiration. Blue light works as a signal to guard cells, to induce intracellular signaling and open stomata. Blue light receptor phototropins (phots) are activated by blue light; phot-mediated signals promote plasma membrane (PM) H+-ATPase activity via C-terminal Thr phosphorylation, serving as the driving force for stomatal opening in guard cells. However, the details of this signaling process are not fully understood. In this study, through an in vitro screening of phot-interacting protein kinases, we obtained the CBC1 and CBC2 that had been reported as signal transducers in stomatal opening. Promoter activities of CBC1 and CBC2 indicated that both genes were expressed in guard cells. Single and double knockout mutants of CBC1 and CBC2 showed no lesions in the context of phot-mediated phototropism, chloroplast movement, or leaf flattening. In contrast, the cbc1cbc2 double mutant showed larger stomatal opening under both dark and blue light conditions. Interestingly, the level of phosphorylation of C-terminal Thr of PM H+-ATPase was higher in double mutant guard cells. The larger stomatal openings of the double mutant were effectively suppressed by the phytohormone abscisic acid (ABA). CBC1 and CBC2 interacted with BLUS1 and PM H+-ATPase in vitro. From these results, we conclude that CBC1 and CBC2 act as negative regulators of stomatal opening, probably via inhibition of PM H+-ATPase activity.

HTLV-1 Tax Induces Formation of the Active Macromolecular IKK Complex by Generating Lys63- and Met1-Linked Hybrid Polyubiquitin Chains.

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is crucial for the development of adult T-cell leukemia (ATL), a highly malignant CD4+ T cell neoplasm. Among the multiple aberrant Tax-induced effects on cellular processes, persistent activation of transcription factor NF-κB, which is activated only transiently upon physiological stimulation, is essential for leukemogenesis. We and others have shown that Tax induces activation of the IκB kinase (IKK) complex, which is a critical step in NF-κB activation, by generating Lys63-linked polyubiquitin chains. However, the molecular mechanism underlying Tax-induced IKK activation is controversial and not fully understood. Here, we demonstrate that Tax recruits linear (Met1-linked) ubiquitin chain assembly complex (LUBAC) to the IKK complex and that Tax fails to induce IKK activation in cells that lack LUBAC activity. Mass spectrometric analyses revealed that both Lys63-linked and Met1-linked polyubiquitin chains are associated with the IKK complex. Furthermore, treatment of the IKK-associated polyubiquitin chains with Met1-linked-chain-specific deubiquitinase (OTULIN) resulted in the reduction of high molecular weight polyubiquitin chains and the generation of short Lys63-linked ubiquitin chains, indicating that Tax can induce the generation of Lys63- and Met1-linked hybrid polyubiquitin chains. We also demonstrate that Tax induces formation of the active macromolecular IKK complex and that the blocking of Tax-induced polyubiquitin chain synthesis inhibited formation of the macromolecular complex. Taken together, these results lead us to propose a novel model in which the hybrid-chain-dependent oligomerization of the IKK complex triggered by Tax leads to trans-autophosphorylation-mediated IKK activation.

The ubiquitin ligase STUB1 regulates stability and activity of RUNX1 and RUNX1-RUNX1T1.

RUNX1 is a member of RUNX transcription factors and plays important roles in hematopoiesis. Disruption of RUNX1 activity has been implicated in the development of hematopoietic neoplasms. Chromosomal translocations involving the RUNX1 gene are associated with several types of leukemia, including acute myeloid leukemia driven by a leukemogenic fusion protein RUNX1-RUNX1T1. Previous studies have shown that RUNX1 is an unstable protein and is subjected to proteolytic degradation mediated by the ubiquitin-proteasome pathway. However, the precise mechanisms of RUNX1 ubiquitination have not been fully understood. Furthermore, much less is known about the mechanisms to regulate the stability of RUNX1-RUNX1T1. In this study, we identified several RUNX1-interacting E3 ubiquitin ligases using a novel high-throughput binding assay. Among them, we found that STUB1 bound to RUNX1 and induced its ubiquitination and degradation mainly in the nucleus. Immunofluorescence analyses revealed that the STUB1-induced ubiquitination also promoted nuclear export of RUNX1, which probably contributes to the reduced transcriptional activity of RUNX1 in STUB1-overexpressing cells. STUB1 also induced ubiquitination of RUNX1-RUNX1T1 and down-regulated its expression. Importantly, STUB1 overexpression showed a substantial growth-inhibitory effect in myeloid leukemia cells that harbor RUNX1-RUNX1T1, whereas it showed only a marginal effect in other non-RUNX1-RUNX1T1 leukemia cells and normal human cord blood cells. Taken together, these data suggest that the E3 ubiquitin ligase STUB1 is a negative regulator of both RUNX1 and RUNX1-RUNX1T1. Activation of STUB1 could be a promising therapeutic strategy for RUNX1-RUNX1T1 leukemia.

The ubiquitin ligase RNF38 promotes RUNX1 ubiquitination and enhances RUNX1-mediated suppression of erythroid transcription program.

RUNX1 is a member of RUNX transcription factors and plays important roles in hematopoiesis. RUNX1 function is under the tight control through posttranslational modifications, including phosphorylation and ubiquitination. We previously developed a luminescence-based binding assay (AlphaScreen) to systematically detect RUNX1-interacting E3 ubiquitin ligases. In this study, we showed that a nuclear ubiquitin ligase RNF38 induced ubiquitination of RUNX1. RNF38-induced RUNX1 ubiquitination did not promote RUNX1 degradation, but rather stabilized RUNX1 protein. We also found that RNF38 enhanced RUNX1-mediated transcriptional repression of the erythroid master regulator KLF1 in K562 cells. Consequently, RNF38 cooperated with RUNX1 to inhibit erythroid differentiation of K562 cells. Thus, our study identified RNF38 as a novel E3 ligase that modifies RUNX1 function without inducing its degradation.

DANFIN functions as an inhibitor of transcription factor NF-κB and potentiates the antitumor effect of bortezomib in multiple myeloma.

Nuclear factor-κB (NF-κB) proteins are transcription factors that play key roles in regulating most immune responses and cell death. Constitutively active NF-κB has been shown to exhibit chemoresistance by inducing anti-apoptosis in tumor cells. Multiple myeloma is known as a constitutive NF-κB activating disease, and the proteasome inhibitor bortezomib is used to treat multiple myeloma and mantle cell lymphoma. We demonstrate here that DANFIN (N,N'-bis-(2,4-dimethyl-phenyl)-ethane-1,2-diamine) functions as an inhibitor of the p65 family proteins and induces chemosensitization to bortezomib in multiple myeloma. DANFIN was found to be an inhibitor of interactions between p65 and IκBα without the inhibition of the DNA binding activity of the p65 protein. In addition, DANFIN affected the IκBα binding region in Rel Homology Domain (RHD) and suppressed the nuclear translocalization of the p65 protein in cells. Furthermore, in multiple myeloma cells, DANFIN suppressed the expression level of NF-κB target genes and induced apoptosis. The combination therapy of DANFIN with bortezomib dramatically enhanced the apoptosis of multiple myeloma cells and indicated a remarkable anti-tumor effect in a multiple-myeloma xenograft mouse model.

Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication.

Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells.

Synthesis and Characterization of the First Borosulfates of Magnesium, Manganese, Cobalt, Nickel, and Zinc.

The first magnesium, manganese, cobalt, nickel, and zinc borosulfates were synthesized employing solvothermal conditions starting from the superacid H[B(HSO4)4] and the respective metal powders (Mg, Ni, Zn) or oxides (MnO2, CoO). α- M4[B2O(SO4)6] ( M = Mg, Mn, Co, Ni, Zn) crystallize isotypically in a new structure type in P3̅ (No. 147) with Z = 1, a = 793.59(4)-810.86(9) pm, and c = 743.98(4)-775.09(9) pm. The oligomeric anion comprises unprecedented dimeric open-branched quadruple tetrahedra { oB, 4 t}[B2O(SO4)6]8-, which are connected via M2O9 dimers to give a three-dimensional network. Upon mild heating, we observed a phase change from α-Mg4[B2O(SO4)6] to ß-Mg4[B2O(SO4)6], yielding a further new structure type in P3̅ (No. 147) with Z = 3, a = 1391.96(6) pm, and c = 748.54(3) pm. The reaction of MgB2 with SO3 yields Mg[B2(SO4)4] crystallizing in C2/ c with Z = 4, a = 1744.28(10) pm, b = 531.45(3) pm, c = 1429.06(8) pm, and ß = 126.323(2)° showing phyllosilicate topology. UV/vis spectroscopy on α- TM4[B2O(SO4)6] ( TM = Co, Ni) confirms the valence state of the TM and reveals that borosulfates are weakly coordinating host structures. Structure relationships between the presented crystal structures and similar borophosphates are shown. The results of vibrational spectroscopy as well as magnetic and thermal measurement investigations are discussed.

Ensuring safe drug administration to pediatric patients with renal dysfunction: a multicenter study.

BACKGROUND: In pediatric patients, due to variations in baseline serum creatinine (Cr) reference values, renal dysfunctions sometimes go unnoticed. In addition, renally excreted drugs need dose adjustment while nephrotoxic drugs should be avoided altogether in patients with impaired renal function. However, most physicians are apparently unaware of these facts and may administer these drugs to vulnerable patients. METHODS: We administered a questionnaire to all physicians and pharmacists specializing in pediatric medical care at six Tokyo metropolitan government-run hospitals in Japan. RESULTS: 276 (59%) of 470 physicians and pharmacists participated. The rate of correct answers given by physicians who were asked to state the serum Cr reference range for 4-year-olds and 8-year-olds was 83 and 74%, respectively. On the other hand, the rate of correct answers given by pharmacists to the same question was only 27 and 24%, respectively. Only about 50% of physicians were aware that histamine H2-receptor antagonists and oseltamivir are renally excreted or that acyclovir and angiotensin II receptor blocker are nephrotoxic. However, most of the pharmacists recognized that histamine H2-receptor antagonists and oseltamivir are renally excreted drugs. CONCLUSIONS: For the majority of the investigated drugs, the awareness that we need to reduce dosages for patients with renal dysfunction was insufficient. To ensure safe drug administration, communication between physicians and pharmacists is paramount. There is an urgent need for the creation of a safe drug administration protocol for pediatric patients with renal dysfunction.

MyoD reprogramming requires Six1 and Six4 homeoproteins: genome-wide cis-regulatory module analysis.

Myogenic regulatory factors of the MyoD family have the ability to reprogram differentiated cells toward a myogenic fate. In this study, we demonstrate that Six1 or Six4 are required for the reprogramming by MyoD of mouse embryonic fibroblasts (MEFs). Using microarray experiments, we found 761 genes under the control of both Six and MyoD. Using MyoD ChIPseq data and a genome-wide search for Six1/4 MEF3 binding sites, we found significant co-localization of binding sites for MyoD and Six proteins on over a thousand mouse genomic DNA regions. The combination of both datasets yielded 82 genes which are synergistically activated by Six and MyoD, with 96 associated MyoD+MEF3 putative cis-regulatory modules (CRMs). Fourteen out of 19 of the CRMs that we tested demonstrated in Luciferase assays a synergistic action also observed for their cognate gene. We searched putative binding sites on these CRMs using available databases and de novo search of conserved motifs and demonstrated that the Six/MyoD synergistic activation takes place in a feedforward way. It involves the recruitment of these two families of transcription factors to their targets, together with partner transcription factors, encoded by genes that are themselves activated by Six and MyoD, including Mef2, Pbx-Meis and EBF.

Dysregulation of a potassium channel, THIK-1, targeted by caspase-8 accelerates cell shrinkage.

Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the physiological role of CASP8 is not fully understood. Here, we identified a two-pore domain potassium channel, tandem-pore domain halothane-inhibited K+ channel 1 (THIK-1), as a novel CASP8 substrate. The intracellular region of THIK-1 was cleaved by CASP8 in apoptotic cells. Overexpression of THIK-1, but not its mutant lacking the CASP8-target sequence in the intracellular portion, accelerated cell shrinkage in response to apoptotic stimuli. In contrast, knockdown of endogenous THIK-1 by RNA interference resulted in delayed shrinkage and potassium efflux. Furthermore, a truncated THIK-1 mutant lacking the intracellular region, which mimics the form cleaved by CASP8, led to a decrease of cell volume of cultured cells without apoptotic stimulation and excessively promoted irregular development of Xenopus embryos. Taken together, these results indicate that THIK-1 is involved in the acceleration of cell shrinkage. Thus, we have demonstrated a novel physiological role of CASP8: creating a cascade that advances the cell to the next stage in the apoptotic process.

Sedation protocol with fasting and shorter sleep leads to magnetic resonance imaging success.

BACKGROUND: Young children undergoing magnetic resonance imaging (MRI) require sedation. In June 2013, Tokyo Metropolitan Ohtsuka Hospital (TMOH) introduced an oral sedation protocol for young children undergoing MRI; the protocol included instructions on fasting before sedation, and recommended a shorter duration of sleep the night before MRI. We compared the MRI success rate before and after the introduction of this protocol. METHODS: The eligible subjects were children under 3 years old who underwent MRI by appointment at TMOH between October 2012 and March 2014, under sedation with triclofos sodium. All those who underwent MRI in or after June 2013 were enrolled prospectively as a post-protocol group. All patients who underwent MRI before June 2013 were enrolled retrospectively as a pre-protocol group, with data collected from chart review. RESULTS: Seventy-four patients were enrolled in the post-protocol group, and 42 in the pre-protocol group. The MRI success rate was significantly higher in the post-protocol group than in the pre-protocol group (98.7% vs 88.1%), as was the rate of on-time starting of MRI (86.5% vs 71.4%). The post-protocol group woke up earlier on the day of examination (6:18 a.m. vs 6:43 a.m.), resulting in a significantly longer time between awakening and the beginning of sedation (289.8 min vs 265.9 min), and a significantly shorter average duration of sleep on the previous night (504.8 min vs 532.3 min). CONCLUSIONS: Implementation of a hospital-wide sedation protocol for young children undergoing MRI significantly improved the MRI success rate.

Morphology of the fibular insertion of the posterolateral corner and biceps femoris tendon.

PURPOSE: To clarify the fibular head insertion of the fibular collateral ligament (FCL), popliteofibular ligament (PFL), and biceps femoris tendon and related osseous landmarks on three-dimensional (3-D) images. METHODS: Twenty-one non-paired, formalin-fixed human cadaveric knees were evaluated in this study. The fibular head insertions of the FCL, PFL and biceps femoris tendon were identified and marked. 3-D images were created, and the surface area, location, positional relationships, and morphology of the fibular insertions of the FCL, PFL, and biceps femoris tendon and related osseous structures were analysed. RESULTS: The fibular head had a unique pyramidal shape, and the relationships of the fibular insertion of the FCL, PFL, and biceps femoris tendon were consistent. The fibular head consists of three aspects: lateral aspect, posterior aspect, and proximal tibiofibular facet. The insertions of the FCL, PFL, and biceps femoris tendon were attached to the centre from the distal side of the lateral aspects of the fibular head, posterior aspect of the fibular styloid process, and lateral aspect surrounding the FCL, respectively. The mean surface areas of the FCL and PFL fibular insertions were 100.1 ± 29.5 and 18.5 ± 7.2 mm2, respectively. CONCLUSION: This study showed that the relationships between the characteristic features of the fibular head and insertions of the FCL, PFL, and biceps femoris tendon were consistent. The clinical relevance of this study is that it improves understanding of the anatomy of the insertions of the PLC and biceps femoris tendon.