Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates specific downstream protein kinases including CaMKI, CaMKIV and 5'-AMP-activated protein kinase. In order to examine the variety of CaMKK-mediated signaling pathways, we searched for novel CaMKK substrate(s) using N(6)-(1-methylbutyl)-ATP and genetically engineered CaMKKα mutant, CaMKKα (Phe(230)Gly), that was capable of utilizing this ATP analogue as a phosphate donor. Incubation of rat brain extracts with recombinant CaMKKα (Phe(230)Gly), but not with wild-type kinase, in the presence of N(6)-(1-methylbutyl)-ATP and Ca(2+)/CaM, induced significant threonine phosphorylation of a 50kDa protein as well as CaMKI phosphorylation at Thr(177). The 50kDa CaMKK substrate was partially purified by using serial column chromatography, and was identified as Syndapin I by LC-MS/MS analysis. We confirmed that recombinant Syndapin I was phosphorylated by CaMKKα and β isoforms at Thr(355)in vitro. Phosphorylation of HA-Syndapin I at Thr(355) in transfected HeLa cells was significantly induced by co-expression of constitutively active mutants of CaMKK isoforms. This is the first report that CaMKK is capable of phosphorylating a non-kinase substrate suggesting the possibility of CaMKK-mediated novel Ca(2+)-signaling pathways that are independent of downstream protein kinases.

We compared the performance of the 3D-Gene® mutation assay (3D-Gene® KRAS mutation assay kit) with the Scorpion-ARMS (therascreen® KRAS RGQ PCR Kit) and Luminex (MEBGEN™ KRAS kit) assays for the detection of KRAS mutations in formalin-fixed, paraffin-embedded tissue samples from 150 patients diagnosed with colorectal cancer. DNA was extracted from the paraffin-embedded tissue samples with or without macrodissection under hematoxylin and eosin staining and the KRAS mutation status was independently determined using these assays. Discordant results were re-analyzed by Sanger sequencing. Mutation detection analysis was successfully performed in all 150 specimens using the 3D-Gene® mutation assay without an invalid case. The concordance rate between the 3D-Gene® mutation assay and Scorpion-ARMS or Luminex was 98.7% (148/150). KRAS mutations were detected at a frequency of 35.3% (53/150) in colorectal cancer specimens. Three discrepant cases were found between the three assays. Overall, our results demonstrate a high concordance rate of between the 3D-Gene® mutation assay and the two existing in-vitro diagnostics kits. All three assays proved to be validated methods for detecting clinically significant KRAS mutations in paraffin-embedded tissue samples.

Pulmonary emphysema is characterized histologically by destruction of alveolar walls and enlargement of air spaces due to lung epithelial cell apoptosis. Cell adhesion molecule 1 (CADM1) is an immunoglobulin superfamily member expressed in lung epithelial cells. CADM1 generates a membrane-associated C-terminal fragment, αCTF, through A disintegrin- and metalloprotease-10-mediated ectodomain shedding, subsequently releasing the intracellular domain (ICD) through γ-secretase-mediated intramembrane shedding of αCTF. αCTF localizes to mitochondria and induces apoptosis in lung epithelial cells. αCTF contributes to the development and progression of emphysema as a consequence of increased CADM1 ectodomain shedding. The purpose of this study was to examine whether the ICD makes a similar contribution.

Biomarkers for the development of type 2 diabetes (T2D) are useful for prediction and intervention of the disease at earlier stages. In this study, we performed a longitudinal study of changes in metabolites using an animal model of T2D, the spontaneously diabetic Torii (SDT) rat. Fasting plasma samples of SDT and control Sprague-Dawley (SD) rats were collected from 6 to 24 weeks of age, and subjected to gas chromatography-mass spectrometry-based metabolome analysis. Fifty-nine hydrophilic metabolites were detected in plasma samples, including amino acids, carbohydrates, sugars and organic acids. At 12 weeks of age, just before the onset of diabetes in SDT rats, the amounts of nine of these metabolites (asparagine, glutamine, glycerol, kynurenine, mannose, n-alpha-acetyllysine, taurine, threonine, and tryptophan) in SDT rats were significantly different from those in SD rats. In particular, metabolites in the tryptophan metabolism pathway (tryptophan and kynurenine) were decreased in SDT rats at 12 weeks of age and later. The lower tryptophan and kynurenine levels in the prediabetic state and later were further confirmed by a replication study on SDT rats and by a longitudinal study on another animal model of T2D, the Otsuka Long-Evans Tokushima Fatty rat. Our data indicate that tryptophan and its metabolites are potential biomarkers for prediabetes and that tryptophan metabolism may be a potential target of intervention for treatment of the disease.

Clay-size minerals play important roles in terrestrial biogeochemistry and atmospheric physics, but their data have been only partially compiled at global scale. We present a global dataset of clay-size minerals in the topsoil and subsoil at different spatial resolutions. The data of soil clay and its mineralogical composition were gathered through a literature survey and aggregated by soil orders of the Soil Taxonomy for each of the ten groups: gibbsite, kaolinite, illite/mica, smectite, vermiculite, chlorite, iron oxide, quartz, non-crystalline, and others. Using a global soil map, a global dataset of soil clay-size mineral distribution was developed at resolutions of 2' to 2° grid cells. The data uncertainty associated with data variability and assumption was evaluated using a Monte Carlo method, and validity of the clay-size mineral distribution obtained in this study was examined by comparing with other datasets. The global soil clay data offer spatially explicit studies on terrestrial biogeochemical cycles, dust emission to the atmosphere, and other interdisciplinary earth sciences.

Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) acts as a regulatory kinase that phosphorylates and activates multiple downstream kinases including CaMKI, CaMKIV, 5'AMP-activated protein kinase (AMPK) and protein kinase B (PKB), resulting in regulation of wide variety of Ca2+-dependent physiological responses under normal and pathological conditions. CaMKKβ is regulated by Ca2+/calmodulin-binding, autophosphorylation, and transphosphorylation by multiple protein kinases including cAMP-dependent protein kinase (PKA). In this report, we found that phosphorylation of CaMKKβ is dynamically regulated by protein phosphatase/kinase system in HeLa cells. Global phosphoproteomic analysis revealed the constitutive phosphorylation at 8 Ser residues including Ser128, 132, and 136 in the N-terminal regulatory domain of rat CaMKKβ in unstimulated HeLa cells as well as inducible phosphorylation of Thr144 in the cells treated with a phosphatase inhibitor, okadaic acid (OA). Thr144 phosphorylation in CaMKKβ has shown to be rapidly induced by OA treatment in a time- and dose-dependent manner in transfected HeLa cells, indicating that Thr144 in CaMKKβ is maintained unphosphorylated state by protein phosphatase(s). We confirmed that in vitro dephosphorylation of pThr144 in CaMKKβ by protein phosphatase 2A and 1. We also found that the pharmacological inhibition of protein phosphatase(s) significantly induces CaMKKβ-phosphorylating activity (at Thr144) in HeLa cell lysates as well as in intact cells; however, it was unlikely that this activity was catalyzed by previously identified Thr144-kinases, such as AMPK and PKA. Taken together, these results suggest that the phosphorylation and dephosphorylation of Thr144 in CaMKKβ is dynamically regulated by multiple kinases/phosphatases signaling resulting in fine-tuning of the enzymatic property.

Auditory ossicles in the middle ear and bony labyrinth of the inner ear are highly mineralized in adult mammals. Cellular mechanisms underlying formation of dense bone during development are unknown. Here, we found that osteoblast-like cells synthesizing highly mineralized hearing-related bones produce both type I and type II collagens as the bone matrix, while conventional osteoblasts and chondrocytes primarily produce type I and type II collagens, respectively. Furthermore, these osteoblast-like cells were not labeled in a "conventional osteoblast"-specific green fluorescent protein (GFP) mouse line. Type II collagen-producing osteoblast-like cells were not chondrocytes as they express osteocalcin, localize along alizarin-labeled osteoid, and form osteocyte lacunae and canaliculi, as do conventional osteoblasts. Auditory ossicles and the bony labyrinth exhibit not only higher bone matrix mineralization but also a higher degree of apatite orientation than do long bones. Therefore, we conclude that these type II collagen-producing hypermineralizing osteoblasts (termed here auditory osteoblasts) represent a new osteoblast subtype. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Sarcopenia is characterized by the loss of skeletal muscle mass and strength and is associated with poor prognosis in patients with chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS) exposure, a major cause for COPD, induces mitochondrial damage, which has been implicated in sarcopenia pathogenesis. The current study sought to examine the involvement of insufficient Parkin-mediated mitophagy, a mitochondrion-selective autophagy, in the mechanisms by which dysfunctional mitochondria accumulate with excessive reactive oxygen species (ROS) production in the development of COPD-related sarcopenia.

Termites with symbiotic methanogens are a known source of atmospheric methane (CH4), but large uncertainties remain regarding the flux magnitude. This study estimated global termite CH4 emissions using a framework similar to previous studies but with contemporary datasets and a biogeochemical model. The global termite emission in 2020 was estimated as 14.8 ± 6.7 Tg CH4 year-1, mainly from tropical and subtropical ecosystems, indicating a major natural source from upland regions. Uncertainties associated with estimation methods were assessed. The emission during the historical period 1901-2021 was estimated to have increased gradually (+ 0.7 Tg CH4 year-1) as a result of combined influences of elevated CO2 (via vegetation productivity), climatic warming, and land-use change. Future projections using climate and land-use scenarios (shared socioeconomic pathways [ssp] 126 and 585) also showed increasing trends (+ 0.5 to 5.9 Tg CH4 year-1 by 2100). These results suggest the importance of termite emissions in the global CH4 budget and, thus, in climatic prediction and mitigation.

Immune checkpoint inhibitors (ICIs) confer a survival benefit in many cancer types. Given that the survival outcome for cancer of unknown primary site (CUP) remains poor, we investigated the potential of CUP for immunotherapy.

Transforming growth factor, beta (TGFB) signal is considered to be a tumor suppressive pathway based on the frequent genomic deletion of the SMAD4 gene in pancreatic cancer (PC); however; the role of the activin signal, which also belongs to the TGFB superfamily, remains largely unclear.

Cyclin G-associated kinase (GAK), a key player in clathrin-mediated membrane trafficking, is overexpressed in various cancer cells. Here, we report that GAK expression is positively correlated with the Gleason score in surgical specimens from prostate cancer patients. Embryonic fibroblasts from knockout mice expressing a kinase-dead (KD) form of GAK showed constitutive hyper-phosphorylation of the epidermal growth factor receptor (EGFR). In addition to the well-known EGFR inhibitors gefitinib and erlotinib, the dietary flavonoid luteolin was a potent inhibitor of the Ser/Thr kinase activity of GAK in vitro. Co-administration of luteolin and gefitinib to PC-3 cells had a greater effect on cell viability than administration of either compound alone; this decrease in viability was associated with drastic down-regulation of GAK protein expression. A comprehensive microRNA array analysis revealed increased expression of miR-630 and miR-5703 following treatment of PC-3 cells with luteolin and/or gefitinib, and exogenous overexpression of miR-630 caused growth arrest of these cells. GAK appears to be essential for cell death because co-administration of gefitinib and luteolin to EGFR-deficient U2OS osteosarcoma cells also had a greater effect on cell viability than administration of either compound alone. Taken together, these findings suggest that GAK may be a new therapeutic target for prostate cancer and osteosarcoma.

To determine cellular distribution of cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member, in the human oxyntic gastric mucosa, and to explore possible involvement in the development and peritoneal dissemination of signet ring cell (SRC) gastric carcinoma, which often develops in the oxyntic mucosa.

We have recently reported that Ca2+/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) is inactivated by reactive sulfur species via polysulfidation of the active-site Cys residue. Here, we show that hydrogen peroxide (H2O2) limit CaMKIV activity at the same active-site Cys residue through oxidation and downstream signaling in cells. CaMKIV is phosphorylated at Thr196 by its upstream CaMK kinase (CaMKK), which induces its full activity. In vitro incubation of CaMKIV with H2O2 resulted in reversible inhibition of CaMKK-induced phospho-Thr196 and the consequent inactivation of CaMKIV. In contrast, mutated CaMKIV (C198V) was refractory to the H2O2-induced enzyme inhibition. In transfected cells expressing CaMKIV, Ca2+ ionophore-induced CaMKIV phosphorylation at Thr196 was decreased upon treatment with H2O2, whereas cells expressing mutant CaMKIV (C198V) were resistant to H2O2 treatment. Modification of free thiol with N-ethylmaleimide revealed that Cys198 in CaMKIV is a target for S-oxidation. Additionally, the Ca2+ influx-induced phospho-Thr196 of endogenous CaMKIV was also inhibited upon treatment with H2O2 in Jurkat T-lymphocytes and cerebellar granule cells. Phosphorylation of cyclic AMP response element-binding protein (CREB) at Ser133, which is downstream of CaMKIV, was also decreased upon treatment with H2O2. Thus, our results indicate that oxidation stress regulates cellular function by decreasing the activity of CaMKIV through Cys198 oxidation.

Chemokines and their receptors are potential therapeutic targets in rheumatoid arthritis (RA). Among these, several studies suggested the involvement of CXC chemokine 4 (CXCR4) and its ligand CXC ligand 12 (SDF-1) in RA pathogenesis. However, the role of these molecules in T-cell function is not known completely because of embryonic lethality of Cxcr4- and Cxcl12-deficient mice. In this report, we generated T cell-specific Cxcr4-deficient mice and showed that the CXCR4 in T cells is important for the development of collagen-induced arthritis (CIA).

Cell adhesion molecules act as tumor suppressors primarily by cell attachment activity, but additional mechanisms modifying signal transduction are suggested in some cases. Cell adhesion molecule 1 (CADM1), a membrane-spanning immunoglobulin superfamily, mediates intercellular adhesion by trans-homophilic interaction and acts as a tumor suppressor. Here, we investigated CADM1-associated proteins comprehensively using proteomic analysis of immune-precipitates of CADM1 by mass spectrometry and identified a transmembrane adaptor protein, Csk-binding protein (Cbp), known to suppress Src-mediated transformation, as a binding partner of CADM1. CADM1 localizes to detergent-resistant membrane fractions and co-immunoprecipitated with Cbp and c-Src. Suppression of CADM1 expression using siRNA reduces the amount of co-immunoprecipitated c-Src with Cbp and activates c-Src in colon cancer cells expressing both CADM1 and Cbp. On the other hand, co-replacement of CADM1 and Cbp in colon cancer cells lacking CADM1 and Cbp expression suppresses c-Src activation, wound healing and tumorigenicity in nude mice. Furthermore, expression of Cbp and CADM1 was lost in 55% and 83% of human colon cancer, respectively, preferentially in tumors with larger size and/or lymph node metastasis. CADM1 would act as a colon tumor suppressor by intervening oncogenic c-Src signaling through binding with Cbp besides its authentic cell adhesion activity.

Fibroblast growth factor receptor (FGFR) gene alterations are relatively frequent in lung squamous cell carcinoma (LSCC) and are a potential targets for therapy with FGFR inhibitors. However, little is known regarding the clinicopathologic features associated with FGFR alterations. The angiokinase inhibitor nintedanib has shown promising activity in clinical trials for non-small cell lung cancer. We have now applied next-generation sequencing (NGS) to characterize FGFR alterations in LSCC patients as well as examined the antitumor activity of nintedanib in LSCC cell lines positive for FGFR1 copy number gain (CNG). The effects of nintedanib on the proliferation of and FGFR signaling in LSCC cell lines were examined in vitro, and its effects on tumor formation were examined in vivo. A total of 75 clinical LSCC specimens were screened for FGFR alterations by NGS. Nintedanib inhibited the proliferation of FGFR1 CNG-positive LSCC cell lines in association with attenuation of the FGFR1-ERK signaling pathway in vitro and in vivo. FGFR1 CNG (10.7%), FGFR1 mutation (2.7%), FGFR2 mutation (2.7%), FGFR4 mutation (5.3%), and FGFR3 fusion (1.3%) were detected in LSCC specimens by NGS. Clinicopathologic features did not differ between LSCC patients positive or negative for FGFR alterations. However, among the 36 patients with disease recurrence after surgery, prognosis was significantly worse for those harboring FGFR alterations. Screening for FGFR alterations by NGS warrants further study as a means to identify patients with LSCC recurrence after surgery who might benefit from nintedanib therapy.

Gastrointestinal stromal tumor (GIST), the most common mesenchymal tumor of the human gastrointestinal tract, differentiating toward the interstitial cell of Cajal (ICC), arises predominantly in the stomach and small intestine. Small intestinal GISTs appear to have worse prognosis than gastric GISTs. In a pilot study of a cDNA expression chip using several GISTs, we found that Cell Adhesion Molecule 1 (CADM1), which could contribute to tumor growth and infiltration, is expressed more strongly in small intestinal GISTs than gastric GISTs. In the present study, we examined CADM1 expression in GISTs of different sites and with different gene abnormalities using a large number of gastric and small intestinal GISTs. First, immunoblotting confirmed significantly higher CADM1 expression in small intestinal GISTs with exon 11 c-kit mutation than gastric GISTs with exon 11 c-kit mutation. Real-time PCR also revealed that small intestinal GISTs with exon 11 c-kit mutation showed significantly higher CADM1 mRNA than gastric GISTs with exon 11 c-kit mutation. Although most small intestinal GISTs showed high CADM1 mRNA expression regardless of gene abnormality types, different CADM1 expression was detected between gastric GISTs with c-kit mutation and those with PDGFRA mutation. Immunohistochemistry showed that many small intestinal GISTs were CADM1-positive but most gastric GISTs CADM1-negative or -indefinite. In the normal gastric and small intestinal walls, immunoreactivity of CADM1 was detected only in nerves, but neither in gastric ICCs nor small intestinal ICCs, indicating that the high CADM1expression in small intestinal GISTs might be acquired during tumorigenesis. Different CADM1 expression between gastric and small intestinal GISTs might be related to different prognoses between them. Further functional experiments are needed to elucidate the role of CADM1 on GIST biology, and there is a possibility that targeting therapy against CADM1 has a preventive effect for tumor spreading in small intestinal GISTs.

Most biological functions controlled by the brain and their related disorders are closely associated with activation in specific regions of the brain. Neuroproteomics has been applied to the analysis of whole brain, and the general pattern of protein expression in all regions has been elucidated. However, the comprehensive proteome of each brain region remains unclear.

The malignant Hodgkin/Reed-Sternberg (HRS) cells of Hodgkin lymphoma (HL) are believed to derive from germinal center (GC) B cells, but lack expression of a functional B cell receptor. As apoptosis is the normal fate of B-cell receptor-negative GC B cells, mechanisms that abrogate apoptosis are thus critical in HL development, such as epigenetic disruption of certain pro-apoptotic cancer genes including tumor suppressor genes. Identifying methylated genes elucidates oncogenic mechanisms and provides valuable biomarkers; therefore, we performed a chemical epigenetic screening for methylated genes in HL through pharmacological demethylation and expression profiling. IGSF4/CADM1/TSLC1, a pro-apoptotic cell adhesion molecule of the immunoglobulin superfamily, was identified together with other methylated targets. In contrast to its expression in normal GC B cells, IGSF4 was down-regulated and methylated in HL cell lines, most primary HL, and microdissected HRS cells of 3/5 cases, but not in normal peripheral blood mononuclear cells and seldom in normal lymph nodes. We also detected IGSF4 methylation in sera of 14/18 (78%) HL patients but seldom in normal sera. Ectopic IGSF4 expression decreased HL cells survival and increased their sensitivity to apoptosis. IGSF4 induction that normally follows heat shock stress treatment was also abrogated in methylated lymphoma cells. Thus, our data demonstrate that IGSF4 silencing by CpG methylation provides an anti-apoptotic signal to HRS cells important in HL pathogenesis.