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Mitohormesis defines the increase in fitness induced by adaptive responses to mild mitochondrial stress. Here, we show that N-acetyloxfenicine (NAO) exerted higher thermotolerance than an endogenous mitohormesis-inducer, N-acetyltyrosine. This activity was not observed in armyworm larvae injected with oxfenicine, suggesting the importance of N-acetylation. NAO-induced hormetic effect was triggered by transient perturbation of mitochondria, which causes a small increase in ROS production and leads to retrograde responses including enhanced expression of antioxidant enzyme genes via activation of FoxO transcription factors. Furthermore, pretreatment with NAO significantly repressed stress-induced peroxidation of lipids in mice and growth of colorectal cancer HCT116 cells that had been transplanted into nude mice. Taken together, NAO is a potent mitohormesis-inducer that is similar to N-acetyltyrosine in terms of structure and functions.

Supplementary Materials and Methods Supplementary References Figures S1 to S9 Table S1

Background: Early detection is essential to improve the outcome of patients with pancreatic cancer. A noninvasive and cost-effective diagnostic test using plasma/serum biomarkers would facilitate the detection of pancreatic cancer at the early stage.Methods: Using a novel combination of hollow fiber membrane–based low-molecular-weight protein enrichment and LC-MS-based quantitative shotgun proteomics, we compared the plasma proteome between 24 patients with pancreatic cancer and 21 healthy controls (training cohort). An identified biomarker candidate was then subjected to a large blinded independent validation (n = 237, validation cohort) using a high-density reverse-phase protein microarray.Results: Among a total of 53,009 MS peaks, we identified a peptide derived from CXC chemokine ligand 7 (CXCL7) that was significantly reduced in pancreatic cancer patients, showing an area under curve (AUC) value of 0.84 and a P value of 0.00005 (Mann–Whitney U test). Reduction of the CXCL7 protein was consistently observed in pancreatic cancer patients including those with stage I and II disease in the validation cohort (P < 0.0001). The plasma level of CXCL7 was independent from that of CA19-9 (Pearson's r = 0.289), and combination with CXCL7 significantly improved the AUC value of CA19-9 to 0.961 (P = 0.002).Conclusions: We identified a significant decrease of the plasma CXCL7 level in patients with pancreatic cancer, and combination of CA19-9 with CXCL7 improved the discriminatory power of the former for pancreatic cancer.Impact: The present findings may provide a new diagnostic option for pancreatic cancer and facilitate early detection of the disease. Cancer Epidemiol Biomarkers Prev; 20(1); 160–71. ©2011 AACR.

Supplementary Figures 1-3 from Reduced Plasma Level of CXC Chemokine Ligand 7 in Patients with Pancreatic Cancer

Supplementary Tables 1-2 from Reduced Plasma Level of CXC Chemokine Ligand 7 in Patients with Pancreatic Cancer

Purpose: An invasive growth pattern is one of the hallmarks of pancreatic ductal carcinoma. Actinin-4 is an actin-binding protein associated with enhanced cell motility, invasive growth, and lymph node metastasis. Actinin-4 might play an important role in the development and progression of pancreatic cancer.Experimental Design: The expression of actinin-4 was examined immunohistochemically in 173 cases of invasive pancreatic ductal carcinoma. The copy number of the actinin-4 (ACTN4) gene was calculated by fluorescence in situ hybridization. The expression of actinin-4 was stably knocked down by short hairpin RNA, and tumorigenicity was evaluated by orthotopic implantation into mice with severe combined immunodeficiency.Results: The expression level of actinin-4 was increased in 109 (63.0%) of 173 cases of pancreatic cancer. Kaplan-Meier survival curves revealed that patients with increased expression of actinin-4 had a significantly poorer outcome (P = 0.00001, log-rank test). Multivariate analysis by the Cox proportional hazard model showed that high expression of actinin-4 was the most significant independent negative predictor of survival (hazard ratio, 2.33; P = 0.000009). Amplification (defined as more than four copies per interphase nucleus) of the ACTN4 gene was detected in 11 (37.9%) of 29 cases showing increased expression of actinin-4. Knockdown of actinin-4 expression inhibited the destructive growth of cancer cells in the pancreatic parenchyma.Conclusion: Recurrent amplification of chromosome 19q13.1-2 has been reported in pancreatic cancer, but the exact target gene has not been identified. Actinin-4 contributes to the invasive growth of pancreatic ductal carcinoma, and ACTN4 is one of the candidate oncogenes in this chromosome locus.

Background: The aim of this study was to identify a new plasma biomarker for use in early detection of colorectal cancer.Methods: Using the combination of hollow fiber membrane (HFM)-based low-molecular weight protein enrichment and two-dimensional image converted analysis of liquid chromatography and mass spectrometry (2DICAL), we compared the plasma proteome of 22 colorectal cancer patients with those of 21 healthy controls. An identified biomarker candidate was then validated in two larger cohorts [validation-1 (n = 210) and validation-2 (n = 113)] using a high-density reverse-phase protein microarray.Results: From a total of 53,009 mass peaks, we identified 103 with an area under curve (AUC) value of 0.80 or higher that could distinguish cancer patients from healthy controls. A peak that increased in colorectal cancer patients, with an AUC of 0.81 and P value of 0.0004 (Mann–Whitney U test), was identified as a product of the PLIN2 gene [also known as perilipin-2, adipose differentiation-related protein (ADRP), or adipophilin]. An increase in plasma adipophilin was consistently observed in colorectal cancer patients, including those with stage I or stage II disease (P < 0.0001, Welch's t test). Immunohistochemical analysis revealed that adipophilin is expressed primarily in the basal sides of colorectal cancer cells forming polarized tubular structures, and that it is absent from adjacent normal intestinal mucosae.Conclusions: Adipophilin is a plasma biomarker potentially useful for the detection of early-stage colorectal cancer.Impact: The combination of HFM and 2DICAL enables the comprehensive analysis of plasma proteins and is ideal for use in all biomarker discovery studies. Cancer Epidemiol Biomarkers Prev; 20(10); 2195–203. ©2011 AACR.

Supplementary Data from Expression and Gene Amplification of Actinin-4 in Invasive Ductal Carcinoma of the Pancreas

Expression of human protein kinase C delta (PKCδ) protein has been linked to many types of cancers. PKCδ is known to be a multifunctional PKC family member and has been rigorously studied as an intracellular signaling molecule. Here we show that PKCδ is a secretory protein that regulates cell growth of liver cancer. Full-length PKCδ was secreted to the extracellular space in living liver cancer cells under normal cell culture conditions and in xenograft mouse models. Patients with liver cancer showed higher levels of serum PKCδ than patients with chronic hepatitis or liver cirrhosis or healthy individuals. In liver cancer cells, PKCδ secretion was executed in an endoplasmic reticulum (ER)-Golgi–independent manner, and the inactivation status of cytosolic PKCδ was required for its secretion. Furthermore, colocalization studies showed that extracellular PKCδ was anchored on the cell surface of liver cancer cells via association with glypican 3, a liver cancer–related heparan sulfate proteoglycan. Addition of exogenous PKCδ activated IGF-1 receptor (IGF1R) activation and subsequently enhanced activation of ERK1/2, which led to accelerated cell growth in liver cancer cells. Conversely, treatment with anti-PKCδ antibody attenuated activation of both IGF1R and ERK1/2 and reduced cell proliferation and spheroid formation of liver cancer cells and tumor growth in xenograft mouse models. This study demonstrates the presence of PKCδ at the extracellular space and the function of PKCδ as a growth factor and provides a rationale for the extracellular PKCδ-targeting therapy of liver cancer.Significance:PKCδ secretion from liver cancer cells behaves as a humoral growth factor that contributes to cell growth via activation of proliferative signaling molecules, which may be potential diagnostic or therapeutic targets.

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PDF file - 1.2MB, (A) FACS profiles of KMRC-1 (p53 wildtype) cells that were treated with siDDX31 or siEGFP. Cells were harvested with supernatant, stained with propidium iodide and analyzed by flow cytometry at 96 h after transfection. (B) Effects of DDX31 knockdown on cell growth and p53 and/or p21 expression in p53 mutant cell. Quantitative RT-PCR showing the suppression of endogenous DDX31 expression by DDX31 siRNA oligonucleotides (left). MTT assay showed a slight decrease in the cell numbers upon DDX31 knockdown in KMRC-20 (right ).(C) Effects of DDX31 knockdown on p53 and p21 protein levels in KMRC-20

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PDF file - 1.5MB, (A) Effects of siRNA-mediated NPM1 knockdown on DDX31 localization in the RCC cell line, Caki-1. NPM1 knockdown did not affect DDX31 nucleolar localization. (B) Effects of DDX31 knockdown on Nucleolin localization. Nucleolin localization was not affected by the presence or the absence of DDX31. Scale bar shows 50 mum

PDF file - 1MB, Effects of DDX31 knockdown on p53 and p21 expression at the mRNA and protein levels. The cells were harvested and examined by quantitative RT-PCR at 48h (A) and 72h after transfection (B). (C) western blot analysis with the indicated antibodies at 72h after transfection

PDF file - 1.7MB, S1 (A) western blot analysis of DDX31 transfected HEK293 cells (left panel) and endogenous DDX31 in Caki-1 cells (right panel) using purified anti-DDX31 polyclonal antibody. (B)NPM1 protein expression in RCC and HK-2 cells. (C) NPM1 expression in clinical RCC cases by quantitative RT-PCR. (D) immunohistochemical analysis of NPM1 in ccRCC tissue sections. Representative images of ccRCC tissue (776T) and normal kidney tissue (776N). Scale bar shows 50 mum

PDF file - 1.6MB, (A) Effects of doxorubicin-induced cellular stress. NPM1 translocated to the nucleoplasm or cytoplasm in DDX31-depleted RCC cells, but not in siEGFP-transfected cells even under doxorubicin treatment. �iB) NPM1 translocated to nucleoplasm in HK-2 cells under doxorubicin treatment. Scale bar shows 50 mum

Protein secretion in cancer cells defines tumor survival and progression by orchestrating the microenvironment. Studies suggest the occurrence of active secretion of cytosolic proteins in liver cancer and their involvement in tumorigenesis. Here, we investigated the identification of extended-synaptotagmin 1 (E-Syt1), an endoplasmic reticulum (ER)-bound protein, as a key mediator for cytosolic protein secretion at the ER-plasma membrane (PM) contact sites. Cytosolic proteins interacted with E-Syt1 on the ER, and then localized spatially inside SEC22B

Previous studies reported the critical role of the brefeldin A–inhibited guanine nucleotide exchange protein 3–prohibitin 2 (BIG3‐PHB2) complex in modulating estrogen signaling activation in breast cancer cells, yet its pathophysiological roles in osteosarcoma (OS) cells remain elusive. Here, we report a novel function of BIG3‐PHB2 in OS malignancy. BIG3‐PHB2 complexes were localized mainly in mitochondria in OS cells, unlike in estrogen‐dependent breast cancer cells. Depletion of endogenous BIG3 expression by small interfering RNA (siRNA) treatment led to significant inhibition of OS cell growth. Disruption of BIG3‐PHB2 complex formation by treatment with specific peptide inhibitor also resulted in significant dose‐dependent suppression of OS cell growth, migration, and invasion resulting from G2/M‐phase arrest and in PARP cleavage, ultimately leading to PARP‐1/apoptosis‐inducing factor (AIF) pathway activation–dependent apoptosis in OS cells. Subsequent proteomic and bioinformatic pathway analyses revealed that disruption of the BIG3‐PHB2 complex might lead to downregulation of inner mitochondrial membrane protein complex activity. Our findings indicate that the mitochondrial BIG3‐PHB2 complex might regulate PARP‐1/AIF pathway–dependent apoptosis during OS cell proliferation and progression and that disruption of this complex may be a promising therapeutic strategy for OS., In this study, we focused on understanding the critical role of the mitochondrial BIG3‐PHB2 complex in osteosarcoma (OS) cell proliferation and survival. Its complex disruption by the specific dominant‐peptide inhibitor causes mitochondrial dysfunction, resulting in apoptotic cell death and decreases in the migration and invasion abilities of OS cells. These findings suggest that inhibiting the BIG3‐PHB2 complex formation may be a new therapeutic strategy for the treatment of OS.

Protein secretion in cancer cells defines tumor survival and progression by orchestrating the microenvironment. Studies suggest the occurrence of active secretion of cytosolic proteins in liver cancer and their involvement in tumorigenesis. Here, we investigated the identification of extended-synaptotagmin 1 (E-Syt1), an endoplasmic reticulum (ER)-bound protein, as a key mediator for cytosolic protein secretion at the ER–plasma membrane (PM) contact sites. Cytosolic proteins interacted with E-Syt1 on the ER, and then localized spatially inside SEC22B+vesicles of liver cancer cells. Consequently, SEC22B on the vesicle tethered to the PM via Q-SNAREs (SNAP23, SNX3, and SNX4) for their secretion. Furthermore, inhibiting the interaction of protein kinase Cδ (PKCδ), a liver cancer-specific secretory cytosolic protein, with E-Syt1 by a PKCδ antibody, decreased in both PKCδ secretion and tumorigenicity. Results reveal the role of ER–PM contact sites in cytosolic protein secretion and provide a basis for ER-targeting therapy for liver cancer.

Immunotherapies that target programmed cell death protein 1 (PD-1) and its ligand PD-L1 as well as cytotoxic T-lymphocyte-associated protein 4 (CTLA4) have shown impressive clinical outcomes for multiple tumours. However, only a subset of patients achieves durable responses, suggesting that the mechanisms of the immune checkpoint pathways are not completely understood. Here, we report that PD-L1 translocates from the plasma membrane into the nucleus through interactions with components of the endocytosis and nucleocytoplasmic transport pathways, regulated by p300-mediated acetylation and HDAC2-dependent deacetylation of PD-L1. Moreover, PD-L1 deficiency leads to compromised expression of multiple immune-response-related genes. Genetically or pharmacologically modulating PD-L1 acetylation blocks its nuclear translocation, reprograms the expression of immune-response-related genes and, as a consequence, enhances the anti-tumour response to PD-1 blockade. Thus, our results reveal an acetylation-dependent regulation of PD-L1 nuclear localization that governs immune-response gene expression, and thereby advocate targeting PD-L1 translocation to enhance the efficacy of PD-1/PD-L1 blockade.

Encapsulation is an effective cellular immunity feature in insects, but the mechanism by which hemocytes recognize and encapsulate invading nematodes remains unclear. In addition, insect susceptibility to nematodes differs, indicating differences in immunity. We compared cellular encapsulation with non-parasitic and parasitic nematodes in two lepidopteran insects in vivo and ex vivo. The percentage of the free-living nematode Caenorhabditis elegans (Maupas) (Rhabditida: Rhabditidae) encapsulated following injection into Galleria mellonella Linnaeus (Lepidoptera: Pyralidae) larvae in vivo was low, whereas most larvae were encapsulated in Mythimna separata walker (Lepidoptera: Noctuidae). Similarly, the entomopathogenic nematode Steinernema carpocapsae (Rhabditida: Steinernematidae) was rarely encapsulated in G. mellonella, but > 50% were encapsulated in M. separata. Adhesion of G. mellonella hemocytes on the surface of live C. elegans was infrequently observed ex vivo, whereas dead nematodes were partially covered with hemocytes. A significantly higher percentage of live nematodes was covered with hemocytes in M. separata in the presence and absence of insect plasma compared with G. mellonella. These results indicate that there is a difference in immunity against nematodes between the two insects and that the difference largely depends on the capabilities of the hemocytes.

B-Cell leukemia/lymphoma 11B (BCL11B) is a transcription factor important for T-cell development and acts as a tumor suppressor gene in T-cell acute lymphoblastic leukemia. Here, we identified BCL11B as a candidate leukemia-associated gene in human T-cell leukemia virus type 1 (HTLV-1)-induced adult T-cell leukemia/lymphoma (ATLL). Interestingly, the short form lacking exon 3 (BCL11B/S) protein was more highly expressed than the full-length BCL11B (BCL11B/L) in leukemic cells from most of the ATLL patients, although expression ratios of BCL11B/L to BCL11B/S were almost equal in control CD4

Purpose Glioblastoma (GBM) has a high risk of recurrence and a poor prognosis due to the difficulty of surgical resection and the resistance to temozolomide, the standard treatment for GBM. Therefore, the development of new therapeutic agents for GBM is desired. We searched our compound library for compounds with anti-tumor activity against GBM and identified Curcumin (Cur) derivatives, Compound A and B. The purpose of this study was to investigate the antitumor activity of Compound A and B against GBM. Methods and Results To evaluate the antitumor activity of Compound A and B against GBM, we performed the MTT assay. Human glioblastoma cell lines, U87-MG and U251 cells, were treated with Compound A and B. After 96 hours, cell viability was evaluated using CCK-8, and the IC50 values of Compound A and B were calculated. In U87-MG, the IC50 of Cur was 9.78 μM, whereas the that of Compound A was 2.42 μM and that of Compound B was 1.28 μM. In U251 cells, the IC50 of Cur was 9.50 μM, whereas that of Compound A was 2.27 μM and that of Compound B was 0.64 μM. Next, to examine the effects of Compound A and B on normal cells, we performed the same MTT assay using primary cultured rat astrocytes. At the concentrations at which antitumor effects were observed (Compound A; 3 μM, B; 1.5 μM), there was no reduction in cell viability in primary rat cultured astrocytes. Discussion The present study shows that Compound A and B exhibit antitumor activity against human glioblastoma cells at lower concentrations than Cur without affecting normal cells. We will examine the effects of Compound A and B in a mouse model of brain tumor transplanted with U87-RFP cells in the future, which may lead to the development of noble brain tumor therapeutics.

Purpose Malignant neoplasms arising in the brain and central nervous system have a poor prognosis and present with symptoms such as headache, epileptic seizures, and paralysis of the arms and legs. Treatment of glioma, the most frequent primary brain tumor, is based on surgical removal of the tumor, radiation therapy, and chemotherapy. However, since gliomas grow invasively, surgical removal of the entire tumor is considered difficult. In addition, the standard treatment, temozolomide, possesses the problem of resistance. Therefore, the risk of recurrence is high, and the development of noble therapeutic agents for Glioma is required. We have screened for compounds with anti-tumor activity against Glioma from our natural compound library and focused on ginger extract, Compound A. In this study, we investigated the effect of Compound A on Glioma cell lines. Methods and Results To evaluate the antitumor activity of Compound A on Glioma in vitro , MTT assay was performed. Human glioma cell lines U87-MG and U251 cells were treated with Compound A. After 96 hours, cell viability was evaluated using CCK-8. The IC50 value of Compound A in U87-MG cells was calculated to be 16.3 μM. The IC50 of Compound A in U251 cells was 10.8 μM. Next, to examine the effect of Compound A on normal cells, we performed the same MTT assay using primary cultured rat astrocytes. The MTT assay showed no decrease in cell viability in primary cultured rat astrocytes at 15 μM, whereas an antitumor effect was observed in a human glioma cell line. Discussion In this study, Compound A showed antitumor activity against Glioma without affecting normal cells. In the future, we will examine the effects of Compound A in an immunocompromised mouse brain in which U87-MG-RFP cells are implanted, which may lead to the development of therapeutic agents against Glioma.

Simple Summary The coupling of metabolism with cellular status is critically important and highly evolutionarily conserved. However, how cells coordinate metabolism with transcription as they change their status is not clear. Utilizing multiomic and functional studies, we now demonstrate the dichotomous roles of the Kat3 coactivators CBP and p300 and, in particular, their extreme N-termini, in coordinating cellular metabolism with cell differentiation. Using multiple in vitro and in vivo systems, our study sheds new light on metabolic regulation in homeostasis and disease, including cancer. Abstract The integration of cellular status with metabolism is critically important and the coupling of energy production and cellular function is highly evolutionarily conserved. This has been demonstrated in stem cell biology, organismal, cellular and tissue differentiation and in immune cell biology. However, a molecular mechanism delineating how cells coordinate and couple metabolism with transcription as they navigate quiescence, growth, proliferation, differentiation and migration remains in its infancy. The extreme N-termini of the Kat3 coactivator family members, CBP and p300, by far the least homologous regions with only 66% identity, interact with members of the nuclear receptor family, interferon activated Stat1 and transcriptionally competent β-catenin, a critical component of the Wnt signaling pathway. We now wish to report based on multiomic and functional investigations, utilizing p300 knockdown, N-terminal p300 edited and p300 S89A edited cell lines and p300 S89A knockin mice, that the N-termini of the Kat3 coactivators provide a highly evolutionarily conserved hub to integrate multiple signaling cascades to coordinate cellular metabolism with the regulation of cellular status and function.

Insect parasitic nematodes have acquired mechanisms to evade their host immune response for successful parasitism. Despite the importance of understanding of the evolution of evasion mechanisms from host immunity, insect immune response against non-parasitic nematodes has not been well studied. In our previous study, we demonstrated that a non-insect parasitic nematodeCaenorhabditis eleganswas not encapsulated by haemocytes in the larvae of the greater wax mothGalleria mellonella. To understand how nematodes influence insect haemocytes to escape encapsulation, we examined the effect ofC. eleganson haemocytes in the haemocoel ofG. mellonellalarvae. Injection of nematodes resulted in the decrease of haemocyte density while mortality and spreading ability of haemocytes, the haematopoietic organs were not affected.In vitroco-incubation of haemocytes with nematodes resulted in a decrease of haemocyte density and we observed feeding on haemocytes by nematodes. Injection ofC. elegansfeeding-delay mutants into insects did not cause the decrease of haemocyte density. The decrease of haemocyte density was due to the nematode's ingestion of haemocytes. Furthermore, an entomopathogenic nematode and other bacterial feeding nematodes also showed similar feeding behaviour. The nematode's ability to feed on haemocytes may have played an important role in the evolution of nematode parasitism in bacterial-feeding nematodes.

Recognition of foreign targets by insect hemocytes is a crucial first step for insect immunity against invading multicellular organisms in the hemocoel. To understand the mechanism of recognition, we observed the hemocyte behavior of Galleria mellonella Linnaeus (Lepidoptera: Pyralidae) larvae against beads and the nonparasitic nematode Caenorhabditis elegans (Maupas) (Rhabditida: Rhabditidae) in the presence of plasma ex vivo using time-lapse microscopy. Both granular cells and plasmatocytes adhered to and spread on the surface of beads and nematodes. In addition, the spread plasmatocytes actively moved over the beads and nematodes. These results suggest that not only granular cells but also plasmatocytes can recognize foreign targets in the presence of insect plasma and that spread plasmatocytes can actively search for foreign targets. Hemocyte adhesion to beads and nematodes ex vivo was similar to that of the in vivo 1 h after injection. A divalent cation chelator inhibited the spreading and adhesion of plasmatocytes ex vivo, but it did not affect the adhesion of granular cells. The present method enables the analysis of acute hemocyte response against foreign targets in the presence of plasma.

Immunity to microbial infections is well understood; however, information regarding the immunity to parasitic multicellular organisms remains lacking. To understand innate host cellular immunity to nematodes, we compared the cellular response of the greater wax moth (Galleria mellonella) larvae against the non-parasitic, bacterial-feeding nematodeCaenorhabditis elegansand pathogenic nematodeHeterorhabditis bacteriophora. When intact first-instar or dauer larvae ofC. eleganswere injected into aG. mellonellalarva, most of the nematodes were alive and not confined by the surrounding reaction by insect haemocytes (encapsulation), similarly as the pathogenic nematode, whereas most of the heat-killed nematodes of both species were severely encapsulated by 24 h after inoculation. Other non-parasitic nematodes were also not encapsulated. Surprisingly,C. elegansinjected into the insect haemocoel grew and propagated in the live insect, resulting in death of the host insect. Our results suggest thatC. eleganshas some basic mechanisms to evade immunity ofG. mellonenllaand grow in the haemocoel.

Simple Summary Given their high degree of identity and even greater similarity at the amino acid level, Kat3 coactivators, CBP (Kat3A) and p300 (Kat3B), have long been considered redundant. We describe the generation of novel p300 S89A knock-in mice carrying a single site directed amino acid mutation in p300, changing the highly evolutionarily conserved serine 89 to alanine, thus enhancing Wnt/CBP/catenin signaling (at the expense of Wnt/p300/catenin signaling). p300 S89A knock-in mice exhibit multiple organ system, immunologic and metabolic differences, compared with their wild type counterparts. In particular, these p300 S89A knock-in mice are highly sensitive to intestinal injury resulting in colitis which is known to significantly predispose to colorectal cancer. Our results highlight the critical role of this region in p300 as a signaling nexus and provide further evidence that p300 and CBP are non-redundant, playing definite and distinctive roles in development and disease. Abstract Differential usage of Kat3 coactivators, CBP and p300, by β-catenin is a fundamental regulatory mechanism in stem cell maintenance and initiation of differentiation and repair. Based upon our earlier pharmacologic studies, p300 serine 89 (S89) is critical for controlling differential coactivator usage by β-catenin via post-translational phosphorylation in stem/progenitor populations, and appears to be a target for a number of kinase cascades. To further investigate mechanisms of signal integration effected by this domain, we generated p300 S89A knock-in mice. We show that S89A mice are extremely sensitive to intestinal insult resulting in colitis, which is known to significantly increase the risk of developing colorectal cancer. We demonstrate cell intrinsic differences, and microbiome compositional differences and differential immune responses, in intestine of S89A versus wild type mice. Genomic and proteomic analyses reveal pathway differences, including lipid metabolism, oxidative stress response, mitochondrial function and oxidative phosphorylation. The diverse effects on fundamental processes including epithelial differentiation, metabolism, immune response and microbiome colonization, all brought about by a single amino acid modification S89A, highlights the critical role of this region in p300 as a signaling nexus and the rationale for conservation of this residue and surrounding region for hundreds of million years of vertebrate evolution.

Objective The Emergency Severity Index (ESI) is a highly reliable and valid triage scale that is widely used in emergency departments in not only English language regions but also other countries. The Japan Triage and Acuity Scale (JTAS) is frequently used for emergency patients, and the ESI has not been evaluated against the JTAS in Japan. This study aimed to examine the decision accuracy of the ESI for simulated clinical scenarios among nursing specialists in Japan compared with the JTAS. Method A parallel group randomized trial was conducted. In total, 23 JTAS–trained triage nurses from 10 Japanese emergency departments were randomly assigned to the ESI or the JTAS group. Nurses independently assigned triage categories to 80 emergency cases for the assessment of interrater agreement. Results Interrater agreement between the expert and triage nurses was κ = 0.82 (excellent) in the ESI group and κ = 0.74 (substantial) in the JTAS group. In addition, interrater agreement by acuity was level 2 = 0.42 (moderate) in the ESI group and level 2 = 0.31 (fair) in the JTAS group. Interrater agreement for triage decisions was classified in a higher category in the ESI group than in the JTAS Scale group at level 2. Triage decisions based on the ESI in Japan maintained the same level of interrater agreement and sensitivity as those in other countries. Conclusion These findings suggest that the ESI can be introduced in Japan, despite its different emergency medical background compared with other countries.

Stress enhances the concentration of reactive oxygen species (ROS) in animal plasma. Increased ROS alter various physiological functions, such as development and the immune response, but excessive increases could be harmful. In this study, we tested the hypothesis that abnormally increased plasma ROS levels are associated with animal death. Injection of the nematode Caenorhabditis elegans into insect larvae caused high mortality in Galleria mellonella, and the plasma ROS concentration was four times higher than M9 buffer-injected larvae. There was no difference in plasma antioxidant activity after nematode injection. However, coinjecting nematodes with an antioxidant (ascorbic acid or N-acetylcysteine) suppressed increases in ROS concentrations by the nematodes and increases in the number of nematodes in the larvae, which increased G. mellonella survival. These results suggest that the abnormal elevation of ROS associated with the stress caused by nematode propagation is lethal for G. mellonella.

Insect parasitic nematodes have developed a mechanism to escape from the cellular immunity of their insect hosts for successful parasitism. However, the detailed mechanism whereby they achieve this remains unclear. In our previous study, we demonstrated that non-parasitic nematodes such as Caenorhabditis elegans potentially have the ability to escape from the cellular immunity of the greater wax moth Galleria mellonella. Here we aimed to clarify the effect of non-parasitic and parasitic nematodes on the spreading of hemocytes-an essential cellular reaction for adhering to a foreign substance -from G. mellonella larvae. The hexane/methanol extract of C. elegans inhibited the spreading of hemocytes. Using 2D-TLC and reversed-phase HPLC, we detected a single peak that inhibited the spreading of hemocytes. In addition, the spreading of hemocytes recovered from C. elegans-injected insects was significantly delayed. Western blotting analysis showed that phosphorylated extracellular signal-regulated protein kinase (ERK) -an essential signaling component for spreading in hemocytes-was decreased by the injection of C. elegans, and that plasma from nematode-injected insects contained the factor that causes the decrease of phosphorylated ERK. We also observed this phenomenon using other non-parasitic and parasitic bacterial-feeding nematodes. These results suggest that the factors inhibiting hemocyte adhesion and delaying the spreading of hemocytes are conserved in bacterial-feeding nematodes and could be a pre-adaptation for parasitism.

Mammalian Hedgehog (Hh) signaling plays key roles in embryogenesis and uniquely requires primary cilia. Functional analyses of several ciliogenesis-related genes led to the discovery of the developmental diseases known as ciliopathies. Hence, identification of mammalian factors that regulate ciliogenesis can provide insight into the molecular mechanisms of embryogenesis and ciliopathy. Here, we demonstrate that DYRK2 acts as a novel mammalian ciliogenesis-related protein kinase. Loss ofDyrk2in mice causes suppression of Hh signaling and results in skeletal abnormalities during in vivo embryogenesis. Deletion ofDyrk2induces abnormal ciliary morphology and trafficking of Hh pathway components. Mechanistically, transcriptome analyses demonstrate down-regulation ofAurkaand other disassembly genes followingDyrk2deletion. Taken together, the present study demonstrates for the first time that DYRK2 controls ciliogenesis and is necessary for Hh signaling during mammalian development.

Expression of human protein kinase C delta (PKCδ) protein has been linked to many types of cancers. PKCδ is known to be a multifunctional PKC family member and has been rigorously studied as an intracellular signaling molecule. Here we show that PKCδ is a secretory protein that regulates cell growth of liver cancer. Full-length PKCδ was secreted to the extracellular space in living liver cancer cells under normal cell culture conditions and in xenograft mouse models. Patients with liver cancer showed higher levels of serum PKCδ than patients with chronic hepatitis or liver cirrhosis or healthy individuals. In liver cancer cells, PKCδ secretion was executed in an endoplasmic reticulum (ER)-Golgi–independent manner, and the inactivation status of cytosolic PKCδ was required for its secretion. Furthermore, colocalization studies showed that extracellular PKCδ was anchored on the cell surface of liver cancer cells via association with glypican 3, a liver cancer–related heparan sulfate proteoglycan. Addition of exogenous PKCδ activated IGF-1 receptor (IGF1R) activation and subsequently enhanced activation of ERK1/2, which led to accelerated cell growth in liver cancer cells. Conversely, treatment with anti-PKCδ antibody attenuated activation of both IGF1R and ERK1/2 and reduced cell proliferation and spheroid formation of liver cancer cells and tumor growth in xenograft mouse models. This study demonstrates the presence of PKCδ at the extracellular space and the function of PKCδ as a growth factor and provides a rationale for the extracellular PKCδ-targeting therapy of liver cancer. Significance: PKCδ secretion from liver cancer cells behaves as a humoral growth factor that contributes to cell growth via activation of proliferative signaling molecules, which may be potential diagnostic or therapeutic targets.

Mutations in JAK2, myeloproliferative leukemia virus (MPL), or calreticulin (CALR) occur in hematopoietic stem cells (HSCs) and are detected in more than 80% of patients with myeloproliferative neoplasms (MPNs). They are thought to play a driver role in MPN pathogenesis via autosomal activation of the JAK-STAT signaling cascade. Mutant CALR binds to MPL, activates downstream MPL signaling cascades, and induces essential thrombocythemia in mice. However, embryonic lethality of Calr-deficient mice precludes determination of a role for CALR in hematopoiesis. To clarify the role of CALR in normal hematopoiesis and MPN pathogenesis, we generated hematopoietic cell-specific Calr-deficient mice. CALR deficiency had little effect on the leukocyte count, hemoglobin levels, or platelet count in peripheral blood. However, Calr-deficient mice showed some hematopoietic properties of MPN, including decreased erythropoiesis and increased myeloid progenitor cells in the bone marrow and extramedullary hematopoiesis in the spleen. Transplantation experiments revealed that Calr haploinsufficiency promoted the self-renewal capacity of HSCs. We generated CALRdel52 mutant transgenic mice with Calr haploinsufficiency as a model that mimics human MPN patients and found that Calr haploinsufficiency restored the self-renewal capacity of HSCs damaged by CALR mutations. Only recipient mice transplanted with Lineage-Sca1+c-kit+ cells harboring both CALR mutation and Calr haploinsufficiency developed MPN in competitive conditions, showing that CALR haploinsufficiency was necessary for the onset of CALR-mutated MPNs.

Sorafenib is a multi-kinase inhibitor that has been proven effective for the treatment of unresectable hepatocellular carcinoma (HCC). However, its precise mechanisms of action and resistance have not been well established. We have developed high-density fluorescence reverse-phase protein arrays and used them to determine the status of 180 phosphorylation sites of signaling molecules in the 120 pathways registered in the NCI-Nature curated database in 23 HCC cell lines. Among the 180 signaling nodes, we found that the level of ribosomal protein S6 phosphorylated at serine residue 235/236 (p-RPS6 S235/236) was most significantly correlated with the resistance of HCC cells to sorafenib. The high expression of p-RPS6 S235/236 was confirmed immunohistochemically in biopsy samples obtained from HCC patients who responded poorly to sorafenib. Sorafenib-resistant HCC cells showed constitutive activation of the mammalian target of rapamycin (mTOR) pathway, but whole-exon sequencing of kinase genes revealed no evident alteration in the pathway. p-RPS6 S235/236 is a potential biomarker that predicts unresponsiveness of HCC to sorafenib. The use of mTOR inhibitors may be considered for the treatment of such tumors.