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Arthropathy is a common complication in haemophilia and decreases quality of life. It has been known that concentrations of β-hydroxybutyrate (BHB) in blood are increased by a ketogenic diet, and elevated levels of circulating BHB restricts the progression of inflammation-mediated joint pathological changes. We hypothesized that elevation of blood BHB concentrations could be effective for reducing the progression of bleeding-induced arthropathy by moderating the inflammatory responses of macrophages. In this study, we investigated whether BHB alleviates the arthropathy caused by repeated intra-articular blood injection in rats. To increase blood BHB levels, rats were fed with ketogenic diet. Repeated intra-articular blood injection induced significant joint swelling, whereas ketogenic diet intake significantly increased blood BHB concentrations and ameliorated the joint swelling. The periarticular tissue-fibrosis observed in the control diet intake group appeared to be significantly alleviated in the ketogenic diet intake group. In addition, the IL-1β, which is involved in the progression of arthropathy, levels in the supernatants of blood-exposed macrophages derived from THP-1 cell line were significantly suppressed by BHB supplementation. In summary, BHB moderated the pathological joint changes caused by intra-articular blood exposure., Competing Interests: Declarations. Competing interests: RK, AS, KT, NM, SH, TS, YN, and MS: members of Medical Biology of Thrombosis and Hemostasis established by Nara Medical University and Chugai Pharmaceutical Co., Ltd.RK, NM, SH, TS, YN, and YY: employees of Chugai Pharmaceutical Co., Ltd.RK, NM, SH, and YY: stock ownership of Chugai Pharmaceutical Co., Ltd.TS, YN, YY, and MS: Patents for inventions relating to products of Chugai Pharmaceutical Co., Ltd.AS: speaker’s bureau from CSL Behring.KT: grants or research support from Japan Blood Products Organization, The Mother and Child Health Foundation and Novo Nordisk Pharma.SM, MT, and SK: no competing interest.MS: representative of Medical Biology of Thrombosis and Hemostasis collaborative research laboratory.; research support from Chugai Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd. and CSL Behring.; honoraria or consultation fees from Chugai Pharmaceutical Co., Ltd.; speaker’s bureau from Chugai Pharmaceutical Co., Ltd., CSL Behring, Sanofi, Bayer, Novo Nordisk Pharma, Takeda Pharmaceutical Co., Ltd., Pfizer and Fujimoto Seiyaku Corp. RK, NM, SH, TS, YN, and YY: employees of Chugai Pharmaceutical Co., Ltd. RK, NM, SH, and YY: stock ownership of Chugai Pharmaceutical Co., Ltd. TS, YN, YY, and MS: Patents for inventions relating to products of Chugai Pharmaceutical Co., Ltd. AS: speaker’s bureau from CSL Behring. KT: grants or research support from Japan Blood Products Organization, The Mother and Child Health Foundation and Novo Nordisk Pharma. SM, MT, and SK: no competing interest. MS: representative of Medical Biology of Thrombosis and Hemostasis collaborative research laboratory.; research support from Chugai Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd. and CSL Behring.; honoraria or consultation fees from Chugai Pharmaceutical Co., Ltd.; speaker’s bureau from Chugai Pharmaceutical Co., Ltd., CSL Behring, Sanofi, Bayer, Novo Nordisk Pharma, Takeda Pharmaceutical Co., Ltd., Pfizer and Fujimoto Seiyaku Corp., (© 2024. The Author(s).) more...

During meiosis, chromosomes with homologous partners undergo synaptonemal complex (SC)-mediated pairing, while the remaining unpaired chromosomes are heterochromatinized through unpaired silencing. Mechanisms underlying homolog recognition during SC formation are still unclear. Here, we show that the Caenorhabditis elegans Argonaute proteins, CSR-1 and its paralog CSR-2, interacting with 22G-RNAs, are required for synaptonemal complex formation with accurate homology. CSR-1 in nuclei and meiotic cohesin, constituting the SC lateral elements, were associated with nonsimple DNA repeats, including minisatellites and transposons, and weakly associated with coding genes. CSR-1-associated CeRep55 minisatellites were expressing 22G-RNAs and long noncoding (lnc) RNAs that colocalized with synaptonemal complexes on paired chromosomes and with cohesin regions of unpaired chromosomes. CeRep55 multilocus deletions reduced the efficiencies of homologous pairing and unpaired silencing, which were supported by the csr-1 activity. Moreover, CSR-1 and CSR-2 were required for proper heterochromatinization of unpaired chromosomes. These findings suggest that CSR-1 and CSR-2 play crucial roles in homology recognition, achieving accurate SC formation between chromosome pairs and condensing unpaired chromosomes by targeting repeat-derived lncRNAs., (© 2023 The Authors.) more...

Andecaliximab (ADX) is a monoclonal antibody that inhibits matrix metalloproteinase 9 (MMP9), an extracellular enzyme involved in matrix remodeling, tumor growth, and metastasis. In preclinical models, MMP9 inhibitors have been shown to enhance the cytotoxic effects of chemotherapeutic agents and to suppress distant metastasis. In this phase Ib, multicenter study, the safety and efficacy of ADX combined with S-1 plus cisplatin (SP) or S-1 plus oxaliplatin (SOX) as a first-line treatment were evaluated in Japanese patients with advanced gastric or gastroesophageal junction (GEJ) adenocarcinoma. ADX was administrated at a dose of 800 mg every 2 weeks for the SP cohort and 1200 mg every three weeks for the SOX cohort. As of December 2019, 16 patients were enrolled (six patients in the SP cohort and 10 patients in the SOX cohort). Peripheral sensory neuropathy (69%), anorexia (63%), nausea (56%), and decreased neutrophil counts (44%) were the most common adverse events (AEs). The grade 3 or higher AEs attributed to ADX were stomatitis and abnormal hepatic function (each one patient) in the SP cohort and decreased neutrophil counts (two patients) in the SOX cohort. The objective response rate in 11 patients with measurable target lesions was 73% (8/11), based on the investigator's evaluation. Median progression-free survival was11.9 months (90% confidence interval, 5.6-16.6), and median overall survival was not reached. In conclusion, ADX combined with S-1 plus platinum demonstrated a manageable safety profile and promising clinical activity in the first-line treatment of patients with advanced gastric or GEJ adenocarcinoma.Clinical Trial Registration information: ClinicalTrials.gov Identifier: NCT02862535 (11/08/2016) and protocol ID: GS-US-296-1884., (© 2022. The Author(s).) more...

The central neural network optimizes avoidance behavior depending on the nociceptive stimulation intensity and is essential for survival. How the property of hub neurons that enables the selection of behaviors is genetically defined is not well understood. We show that the transcription factor unc-130, a human FOXD3/4 ortholog, is required to optimize avoidance behavior depending on stimulus strength in Caenorhabditis elegans. unc-130 is necessary for both ON responses (calcium decreases) and OFF responses (calcium increases) in AIBs, central neurons of avoidance optimization. Ablation of predicted upstream inhibitory neurons reduces the frequency of turn behavior, suggesting that optimization needs both calcium responses. At the molecular level, unc-130 upregulates the expression of at least three genes: nca-2, a homolog of the vertebrate cation leak channel NALCN; glr-1, an AMPA-type glutamate receptor; and eat-4, a hypothetical L-glutamate transmembrane transporter in the central neurons of optimization. unc-130 shows more limited regulation in optimizing behavior than an atonal homolog lin-32, and unc-130 and lin-32 appear to act in parallel molecular pathways. Our findings suggest that unc-130 is required for the establishment of some AIB identities to optimize avoidance behavior., (© 2022. The Author(s).) more...

We have studied current induced magnetization switching in W/CoFeB/MgO based three terminal magnetic tunnel junctions. The switching driven by spin-orbit torque (SOT) is evaluated in the so-called type-Y structure, in which the magnetic easy-axis of the CoFeB layer lies in the film plane and is orthogonal to the current flow. The effective spin Hall angle estimated from the bias field dependence of critical current (I c ) is ~ 0.07. The field and current dependence of the switching probability are studied. The field and DC current induced switching can be described using a model based on thermally assisted magnetization switching. In contrast, the 50 ns long pulse current dependence of the switching probability shows significant deviation from the model, even if contribution from the field-like torque is included. The deviation is particularly evident when the threshold switching current is larger. These results show that conventional thermally assisted magnetization switching model cannot be used to describe SOT induced switching using short current pulses., (© 2021. The Author(s).) more...

Surgeons must visually distinguish soft-tissues, such as nerves, from surrounding anatomy to prevent complications and optimize patient outcomes. An accurate nerve segmentation and analysis tool could provide useful insight for surgical decision-making. Here, we present an end-to-end, automatic deep learning computer vision algorithm to segment and measure nerves. Unlike traditional medical imaging, our unconstrained setup with accessible handheld digital cameras, along with the unstructured open surgery scene, makes this task uniquely challenging. We investigate one common procedure, thyroidectomy, during which surgeons must avoid damaging the recurrent laryngeal nerve (RLN), which is responsible for human speech. We evaluate our segmentation algorithm on a diverse dataset across varied and challenging settings of operating room image capture, and show strong segmentation performance in the optimal image capture condition. This work lays the foundation for future research in real-time tissue discrimination and integration of accessible, intelligent tools into open surgery to provide actionable insights., (© 2021. The Author(s).) more...

With the development of massive parallel sequencing technology, it has become easier to establish new model organisms that are ideally suited to the specific biological phenomena of interest. Considering the history of research using classical model organisms, we believe that the efficient construction and sharing of gene mutation libraries will facilitate the progress of studies using these new model organisms. Using C. elegans, we applied the TMP/UV mutagenesis method to animals lacking function in the DNA damage response genes atm-1 and xpc-1. This method produces genetic mutations three times more efficiently than mutagenesis of wild-type animals. Furthermore, we confirmed that the use of next-generation sequencing and the elimination of false positives through machine learning could automate the process of mutation identification with an accuracy of over 95%. Eventually, we sequenced the whole genomes of 488 strains and isolated 981 novel mutations generated by the present method; these strains have been made available to anyone who wants to use them. Since the targeted DNA damage response genes are well conserved and the mutagens used in this study are also effective in a variety of species, we believe that our method is generally applicable to a wide range of animal species. more...

Non-ampullary duodenal adenocarcinoma (NADC) is extremely rare. Little is known about its clinicopathological and molecular features or its management. Herein we retrospectively analyzed the cases of 32 NADC patients, focusing on microsatellite instability (MSI), genetic mutations, CpG island methylator phenotype (CIMP), and immunostaining including mucin phenotype and PD-L1 expression. The incidence of MSI, KRAS/BRAF/GNAS mutations and CIMP was 51.6%, 34.4%/3.1%/6.5% and 28.1%, respectively. PD-L1 expression was seen in 34.4% of patients. No significant associations between clinicopathological features and KRAS/BRAF/GNAS genetic mutations or CIMP were found. Histologically non-well-differentiated-type NADCs and those in the 1st portion of the duodenum were significantly associated with later stages (stages III-IV) (P = 0.006 and P = 0.003, respectively). Gastric-phenotype NADCs were frequently observed in the 1st portion and in late-stage patients; their cancer cells more frequently expressed PD-L1. Histologically, the non-well-differentiated type was an independent predictor of PD-L1 expression in cancer cells (OR 25.05, P = 0.04) and immune cells (OR 44.14, P = 0.02). Only late-stage disease (HR 12.23, P = 0.01) was a prognostic factor for worse overall survival in a Cox proportional hazards regression model. Our observation of high proportions of MSI and PD-L1 expression may prompt the consideration of immune checkpoint inhibitors as a new treatment option for NADCs. more...

The combined phenotype of thrombocytopenia accompanied by intellectual disability in patients with a de novo heterozygous mutation, i.e., p.Tyr64Cys in CDC42, signifies a clinically recognizable novel syndrome that has been eponymized as "Takenouchi-Kosaki syndrome" (OMIM #616737). In the present study, a detailed phenotypic analysis performed for a total of five patients with Takenouchi-Kosaki syndrome revealed that intellectual disability, macrothrombocytopenia, camptodactyly, structural brain abnormalities with sensorineural deafness, hypothyroidism, and frequent infections comprise the cardinal features of this condition. A morphologic analysis of platelets derived from three affected individuals was performed using electron microscopy. The platelets of the three patients were large and spherical in shape. Furthermore, platelet α-granules were decreased, while vacuoles were increased. We further performed a functional analysis of p.Tyr64Cys in CDC42 through CRISPR/Cas9-mediated gene editing in a Caenorhabditis elegans model. This functional analysis suggested that the mutant allele has hypomorphic effects. Takenouchi-Kosaki syndrome is clinically recognizable by the combined phenotype of intellectual disability, macrothrombocytopenia, camptodactyly, structural brain abnormalities with sensorineural deafness, hypothyroidism, and frequent infections as well as the identification of a heterozygous de novo mutation in CDC42, i.e., p.Tyr64Cys. more...

The radiation-induced bystander effect (RIBE) refers to a unique process in which factors released by irradiated cells or tissues exert effects on other parts of the animal not exposed to radiation, causing genomic instability, stress responses and altered apoptosis or cell proliferation. Although RIBEs have important implications for radioprotection, radiation safety and radiotherapy, the molecular identities of RIBE factors and their mechanisms of action remain poorly understood. Here we use Caenorhabditis elegans as a model in which to study RIBEs, and identify the cysteine protease CPR-4, a homologue of human cathepsin B, as the first RIBE factor in nematodes, to our knowledge. CPR-4 is secreted from animals irradiated with ultraviolet or ionizing gamma rays, and is the major factor in the conditioned medium that leads to the inhibition of cell death and increased embryonic lethality in unirradiated animals. Moreover, CPR-4 causes these effects and stress responses at unexposed sites distal to the irradiated tissue. The activity of CPR-4 is regulated by the p53 homologue CEP-1 in response to radiation, and CPR-4 seems to exert RIBEs by acting through the insulin-like growth factor receptor DAF-2. Our study provides crucial insights into RIBEs, and will facilitate the identification of additional RIBE factors and their mechanisms of action. more...

In order to understand the life cycle of hepatitis B virus (HBV) and to develop efficient anti-HBV drugs, a useful in vitro cell culture system which allows HBV infection and recapitulates virus-host interactions is essential; however, pre-existing in vitro HBV infection models are often problematic. Here, we examined the potential of human induced-pluripotent stem (iPS) cell-derived hepatocyte-like cells (iPS-HLCs) as an in vitro HBV infection model. Expression levels of several genes involved in HBV infection, including the sodium taurocholate cotransporting polypeptide (NTCP) gene, were gradually elevated as the differentiation status of human iPS cells proceeded to iPS-HLCs. The mRNA levels of these genes were comparable between primary human hepatocytes (PHHs) and iPS-HLCs. Following inoculation with HBV, we found significant production of HBV proteins and viral RNAs in iPS-HLCs. The three major forms of the HBV genome were detected in iPS-HLCs by Southern blotting analysis. Anti-HBV agents entecavir and Myrcludex-B, which are a nucleoside analogue reverse transcriptase inhibitor and a synthetic pre-S1 peptide, respectively, significantly inhibited HBV infection in iPS-HLCs. These data demonstrate that iPS-HLCs can be used as a promising in vitro HBV infection model. more...

Voltage control of magnetic anisotropy (VCMA) in magnetic heterostructures is a key technology for achieving energy-efficiency electronic devices with ultralow power consumption. Here, we report the first demonstration of the VCMA effect in novel epitaxial Ru/Co 2 FeAl(CFA)/MgO heterostructures with interfacial perpendicular magnetic anisotropy (PMA). Perpendicularly magnetized tunnel junctions with the structure of Ru/CFA/MgO were fabricated and exhibited an effective voltage control on switching fields for the CFA free layer. Large VCMA coefficients of 108 and 139 fJ/Vm for the CFA film were achieved at room temperature and 4 K, respectively. The interfacial stability in the heterostructure was confirmed by repeating measurements. Temperature dependences of both the interfacial PMA and the VCMA effect were also investigated. It is found that the temperature dependences follow power laws of the saturation magnetization with an exponent of ~2, where the latter is definitely weaker than that of conventional Ta/CoFeB/MgO. The significant VCMA effect observed in this work indicates that the Ru/CFA/MgO heterostructure could be one of the promising candidates for spintronic devices with voltage control. more...

Balancer chromosomes are convenient tools used to maintain lethal mutations in heterozygotes. We established a method for engineering new balancers in C. elegans by using the CRISPR/Cas9 system in a non-homologous end-joining mutant. Our studies will make it easier for researchers to maintain lethal mutations and should provide a path for the development of a system that generates rearrangements at specific sites of interest to model and analyse the mechanisms of action of genes. more...

Depression is known to occur frequently in chronic hepatitis C viral (HCV) patients receiving interferon (IFN)-α therapy. In this study, we investigated whether indoleamine 2,3-dioxygenase1 (IDO1)-mediated tryptophan (TRP) metabolism plays a critical role in depression occurring as a side effect of IFN-α therapy. Increases in serum kynurenine (KYN) and 3-hydroxykynurenine (3-HK) concentrations and in the ratios of KYN/TRP and 3-HK/kynurenic acid (KA) were much larger in depressive HCV patients than in non-depressed patients following therapy. Furthermore, transfection of a plasmid continuously expressing murine IFN-γ into normal mice significantly increased depression-like behavior. IFN-γ gene transfer also resulted in a decrease in serum TRP levels in the mice while KYN and 3-HK levels were significantly increased in both serum and frontal cortex. Genetic deletion of IDO1 in mice abrogated both the increase in depression-like behavior and the elevation in TRP metabolites' levels, and the turnover of serotonin in the frontal cortex after IFN-γ gene transfer. These results indicate that the KYN pathway of IDO1-mediated TRP metabolism plays a critical role in depressive symptoms associated with IFN-α therapy. more...

RNA silencing signals in C. elegans spread among cells, leading to RNAi throughout the body. During systemic spread of RNAi, membrane trafficking is thought to play important roles. Here, we show that RNAi Spreading Defective-3 (rsd-3), which encodes a homolog of epsinR, a conserved ENTH (epsin N-terminal homology) domain protein, generally participates in cellular uptake of silencing RNA. RSD-3 is previously thought to be involved in systemic RNAi only in germ cells, but we isolated several deletion alleles of rsd-3, and found that these mutants are defective in the spread of silencing RNA not only into germ cells but also into somatic cells. RSD-3 is ubiquitously expressed, and intracellularly localized to the trans-Golgi network (TGN) and endosomes. Tissue-specific rescue experiments indicate that RSD-3 is required for importing silencing RNA into cells rather than exporting from cells. Structure/function analysis showed that the ENTH domain alone is sufficient, and membrane association of the ENTH domain is required, for RSD-3 function in systemic RNAi. Our results suggest that endomembrane trafficking through the TGN and endosomes generally plays an important role in cellular uptake of silencing RNA. more...

We have studied the magnetic layer thickness dependence of the orbital magnetic moment in magnetic heterostructures to identify contributions from interfaces. Three different heterostructures, Ta/CoFeB/MgO, Pt/Co/AlOx and Pt/Co/Pt, which possess significant interface contribution to the perpendicular magnetic anisotropy, are studied as model systems. X-ray magnetic circular dichroism spectroscopy is used to evaluate the relative orbital moment, i.e. the ratio of the orbital to spin moments, of the magnetic elements constituting the heterostructures. We find that the relative orbital moment of Co in Pt/Co/Pt remains constant against its thickness whereas the moment increases with decreasing Co layer thickness for Pt/Co/AlOx, suggesting that a non-zero interface orbital moment exists for the latter system. For Ta/CoFeB/MgO, a non-zero interface orbital moment is found only for Fe. X-ray absorption spectra shows that a particular oxidized Co state in Pt/Co/AlOx, absent in other heterosturctures, may give rise to the interface orbital moment in this system. These results show element specific contributions to the interface orbital magnetic moments in ultrathin magnetic heterostructures. more...

Autophagosome formation is promoted by the PI3 kinase complex and negatively regulated by myotubularin phosphatases, indicating that regulation of local phosphatidylinositol 3-phosphate (PtdIns3P) levels is important for this early phase of autophagy. Here, we show that the Caenorhabditis elegans myotubularin phosphatase MTM-3 catalyzes PtdIns3P turnover late in autophagy. MTM-3 acts downstream of the ATG-2/EPG-6 complex and upstream of EPG-5 to promote autophagosome maturation into autolysosomes. MTM-3 is recruited to autophagosomes by PtdIns3P, and loss of MTM-3 causes increased autophagic association of ATG-18 in a PtdIns3P-dependent manner. Our data reveal critical roles of PtdIns3P turnover in autophagosome maturation and/or autolysosome formation., (© 2014 The Authors.) more...

Safe and inexpensive energy storage devices with long cycle lifetimes and high power and energy densities are mandatory for the development of electrical power grids that connect with renewable energy sources. In this study, we demonstrated metal-free aqueous redox capacitors using couples comprising low-molecular-weight organic compounds. In addition to the electric double layer formation, proton insertion/extraction reactions between a couple consisting of inexpensive quinones/hydroquinones contributed to the energy storage. This energy storage mechanism, in which protons are shuttled back and forth between two electrodes upon charge and discharge, can be regarded as a proton rocking-chair system. The fabricated capacitor showed a large capacity (>20 Wh/kg), even in the applied potential range between 0-1 V, and high power capability (>5 A/g). The support of the organic compounds in nanoporous carbon facilitated the efficient use of the organic compounds with a lifetime of thousands of cycles. more...

Glycerol-3-phosphate acyltransferase (GPAT) is involved in the first step in glycerolipid synthesis and is localized in both the endoplasmic reticulum (ER) and mitochondria. To clarify the functional differences between ER-GPAT and mitochondrial (Mt)-GPAT, we generated both GPAT mutants in C. elegans and demonstrated that Mt-GPAT is essential for mitochondrial fusion. Mutation of Mt-GPAT caused excessive mitochondrial fragmentation. The defect was rescued by injection of lysophosphatidic acid (LPA), a direct product of GPAT, and by inhibition of LPA acyltransferase, both of which lead to accumulation of LPA in the cells. Mitochondrial fragmentation in Mt-GPAT mutants was also rescued by inhibition of mitochondrial fission protein DRP-1 and by overexpression of mitochondrial fusion protein FZO-1/mitofusin, suggesting that the fusion/fission balance is affected by Mt-GPAT depletion. Mitochondrial fragmentation was also observed in Mt-GPAT-depleted HeLa cells. A mitochondrial fusion assay using HeLa cells revealed that Mt-GPAT depletion impaired mitochondrial fusion process. We postulate from these results that LPA produced by Mt-GPAT functions not only as a precursor for glycerolipid synthesis but also as an essential factor of mitochondrial fusion. more...

BTBD10, an Akt interactor, activates Akt by decreasing the protein phosphatase 2A-mediated dephosphorylation and inactivation of Akt. Overexpression of BTBD10 suppresses motor neuron death that is induced by a familial amyotrophic lateral sclerosis (ALS)-linked superoxide dismutase 1 (SOD1) mutant, G93A-SOD1 in vitro. In this study, we further investigated the BTBD10-mediated suppression of motor neuron death. We found that the small interfering RNA-mediated inhibition of BTBD10 expression led to the death of cultured motor neurons. In Caenorhabditis elegans (C. elegans), disruption of the btbd-10 gene caused not only loss of neurons, including both motor and touch-receptor neurons, but also a locomotion defect. In addition, we found that the expression of BTBD10 was generally decreased in the motor neurons from patients of sporadic ALS and transgenic mice overexpressing G93A-SOD1 (G93A-SOD1-transgenic mice). Collectively, these results suggest that the reduced expression of BTBD10 leads to motor neuron death both in vitro and in vivo. more...

In Caenorhabditis elegans, apoptosis in germ cells is mediated by the same core apoptotic machinery that controls apoptosis in somatic cells. These include the CED-3 caspase, the CED-3 activator CED-4, and the cell death inhibitor CED-9. However, germline apoptosis also differs from somatic apoptosis in its regulation. We found that CSP-3, a caspase homolog that blocks CED-3 autoactivation and apoptosis in somatic cells, does not affect apoptosis in germ cells. Interestingly, the second C. elegans caspase homolog, CSP-2, shares sequence similarity to both catalytic subunits of the CED-3 caspase, and surprisingly, contains a stretch of sequence that is almost identical to that of CSP-3. Unlike CSP-3 that acts specifically in somatic cells, loss of CSP-2 causes increased apoptosis only in germ cells, suggesting that CSP-2 is a germ cell-specific apoptosis inhibitor. Moreover, like CSP-3, CSP-2 associates with the CED-3 zymogen and inhibits its autoactivation, but does not inhibit CED-4-induced CED-3 activation or the activity of the activated CED-3 protease. Thus, two different C. elegans caspase homologs use the same mechanism to prevent caspase autoactivation and apoptosis in different tissues, suggesting that this could be a generally applicable strategy for regulating caspase activation and apoptosis. more...

Asymmetric division is an important property of stem cells. In Caenorhabditis elegans, the Wnt/beta-catenin asymmetry pathway determines the polarity of most asymmetric divisions. The Wnt signalling components such as beta-catenin localize asymmetrically to the cortex of mother cells to produce two distinct daughter cells. However, the molecular mechanism to polarize them remains to be elucidated. Here, we demonstrate that intracellular phospholipase A(1) (PLA(1)), a poorly characterized lipid-metabolizing enzyme, controls the subcellular localizations of beta-catenin in the terminal asymmetric divisions of epithelial stem cells (seam cells). In mutants of ipla-1, a single C. elegans PLA(1) gene, cortical beta-catenin is delocalized and the asymmetry of cell-fate specification is disrupted in the asymmetric divisions. ipla-1 mutant phenotypes are rescued by expression of ipla-1 in seam cells in a catalytic activity-dependent manner. Furthermore, our genetic screen utilizing ipla-1 mutants reveals that reduction of endosome-to-Golgi retrograde transport in seam cells restores normal subcellular localization of beta-catenin to ipla-1 mutants. We propose that membrane trafficking regulated by ipla-1 provides a mechanism to control the cortical asymmetry of beta-catenin. more...

WNK kinases are a small group of unique serine/threonine protein kinases that are conserved among multicellular organisms. Mutations in WNK1-4 cause pseudohypoaldosteronism type II-a form of hypertension. WNKs have been linked to the STE20 kinases and ion carriers, but the underlying molecular mechanisms by which WNKs regulate cellular processes in whole animals are unknown. The Caenorhabditis elegans WNK-like kinase WNK-1 interacts with and phosphorylates germinal centre kinase (GCK)-3--a STE20-like kinase--which is known to inactivate CLH-3, a CIC chloride channel. The wnk-1 or gck-3 deletion mutation causes an Exc phenotype, a defect in the tubular extension of excretory canals. Expression of the activated form of GCK-3 or the clh-3 deletion mutation can partly suppress wnk-1 or gck-3 defects, respectively. These results indicate that WNK-1 controls the tubular formation of excretory canals by activating GCK-3, resulting in downregulation of CIC channel activity. more...

Rab5, a small guanosine triphosphatase, is known to regulate the tethering and docking reaction leading to SNARE (soluble NSF attachment protein receptors)-mediated fusion between endosomes. However, it is uncertain how the signal of the activated Rab5 protein is transduced by its downstream effectors during endosome fusion. Here, we show that the Sec1/Munc18 gene vps-45 is essential for not only viability and development but also receptor-mediated and fluid-phase endocytosis pathways in Caenorhabditis elegans. We found that VPS-45 interacts with a Rab5 effector, Rabenosyn-5 (RABS-5), and the mutants of both vps-45 and rabs-5 show similar endocytic phenotypes. In the macrophage-like cells of vps-45 and rabs-5 mutants, aberrantly small endosomes were accumulated, and the endosome fusion stimulated by the mutant RAB-5 (Q78L) is suppressed by these mutations. Our results indicate that VPS-45 is a key molecule that functions downstream from RAB-5, cooperating with RABS-5, to regulate the dynamics of the endocytic system in multicellular organisms. more...

The p53 tumor suppressor promotes apoptosis in response to DNA damage. Here we describe the Caenorhabditis elegans gene ced-13, which encodes a conserved BH3-only protein. We show that ced-13 mRNA accumulates following DNA damage, and that this accumulation is dependent on an intact C. elegans cep-1/p53 gene. We demonstrate that CED-13 protein physically interacts with the antiapoptotic Bcl-2-related protein CED-9. Furthermore, overexpression of ced-13 in somatic cells leads to the death of cells that normally survive, and this death requires the core apoptotic pathway of C. elegans. Recent studies have implicated two BH3-only proteins, Noxa and PUMA, in p53-induced apoptosis in mammals. Our studies suggest that in addition to the BH3-only protein EGL-1, CED-13 might also promote apoptosis in the C. elegans germ line in response to p53 activation. We propose that an evolutionarily conserved pathway exists in which p53 promotes cell death by inducing expression of two BH3-only genes. more...

Glycosaminoglycans such as heparan sulphate and chondroitin sulphate are extracellular sugar chains involved in intercellular signalling. Disruptions of genes encoding enzymes that mediate glycosaminoglycan biosynthesis have severe consequences in Drosophila and mice. Mutations in the Drosophila gene sugarless, which encodes a UDP-glucose dehydrogenase, impairs developmental signalling through the Wnt family member Wingless, and signalling by the fibroblast growth factor and Hedgehog pathways. Heparan sulphate is involved in these pathways, but little is known about the involvement of chondroitin. Undersulphated and oversulphated chondroitin sulphate chains have been implicated in other biological processes, however, including adhesion of erythrocytes infected with malaria parasite to human placenta and regulation of neural development. To investigate chondroitin functions, we cloned a chondroitin synthase homologue of Caenorhabditis elegans and depleted expression of its product by RNA-mediated interference and deletion mutagenesis. Here we report that blocking chondroitin synthesis results in cytokinesis defects in early embryogenesis. Reversion of cytokinesis is often observed in chondroitin-depleted embryos, and cell division eventually stops, resulting in early embryonic death. Our findings show that chondroitin is required for embryonic cytokinesis and cell division. more...

Calcium (Ca2+) signals regulate a diverse set of cellular responses, from proliferation to muscular contraction and neuro-endocrine secretion. The ubiquitous Ca2+ sensor, calmodulin (CaM), translates changes in local intracellular Ca2+ concentrations into changes in enzyme activities. Among its targets, the Ca2+/CaM-dependent protein kinases I and IV (CaMKs) are capable of transducing intraneuronal signals, and these kinases are implicated in neuronal gene regulation that mediates synaptic plasticity in mammals. Recently, the cyclic AMP response element binding protein (CREB) has been proposed as a target for a CaMK cascade involving not only CaMKI or CaMKIV, but also an upstream kinase kinase that is also CaM regulated (CaMKK). Here, we report that all components of this pathway are coexpressed in head neurons of Caenorhabditis elegans. Utilizing a transgenic approach to visualize CREB-dependent transcription in vivo, we show that this CaMK cascade regulates CRE-mediated transcription in a subset of head neurons in living nematodes. more...

The UNC-51 serine/threonine kinase of C. elegans plays an essential role in axonal elongation, and unc-51 mutants exhibit uncoordinated movements. We have previously identified mouse and human cDNAs encoding UNC-51-like kinase (ULK1). Here we report the identification and characterization of the second murine member of this kinase family, ULK2. Mouse ULK2 cDNA encodes a putative polypeptide of 1033 aa which has an overall 52% and 33% amino acid identity to ULK1 and UNC-51, respectively. ULKs and UNC-51 share a typical domain structure of an amino-terminal kinase domain, a central proline/serine rich (PS) domain, and a carboxy-terminal (C) domain. Northern blot analysis showed that ULK2 mRNA is widely expressed in adult tissues. In situ hybridization analysis indicated that ULK2 mRNA is ubiquitously localized in premature as well as mature neurons in developing nervous system. ULK2 gene was mapped to mouse chromosome 11B1.3 and rat chromosome 10q23 by FISH. HA-tagged ULK2 expressed in COS7 cells had an apparent molecular size of approximately 150 kDa and was autophosphorylated in vitro. Truncation mutants suggested that the autophosphorylation occurs in the PS domain. Although expression of ULK2 failed to rescue unc-51 mutant of C. elegans, a series of ULK2/UNC-51 chimeric kinases revealed that function of the kinase and PS domains are conserved among species, while the C domain acts in a species-specific manner. These results suggest that ULK2 is involved in a previously uncharacterized signaling pathway in mammalian cells. more...