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Although KRAS protein had been classified as an undruggable target, inhibitors of KRAS G12C mutant protein were recently reported to show clinical efficacy in solid tumors. In our previous report, we identified 1-{2,7-diazaspiro[3.5]non-2-yl}prop-2-en-1-one derivative (1) as a KRAS G12C inhibitor that covalently binds to Cys12 of KRAS G12C protein. Compound 1 exhibited potent cellular pERK inhibition and cell growth inhibition against a KRAS G12C mutation-positive cell line and showed an antitumor effect on subcutaneous administration in an NCI-H1373 (KRAS G12C mutation-positive cell line) xenograft mouse model in a dose-dependent manner. In this report, we further optimized the substituents on the quinazoline scaffold based on the structure-based drug design from the co-crystal structure analysis of compound 1 and KRAS G12C to enhance in vitro activity. As a result, ASP6918 was found to exhibit extremely potent in vitro activity and induce dose-dependent tumor regression in an NCI-H1373 xenograft mouse model after oral administration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Highly glycosylated mucins protect epithelial surfaces from external insults and are related to malignant behaviors of carcinoma cells. However, the importance of carbohydrate chains on mucins in the process of cellular protection is not fully understood. Here, we investigated the effect of human mucin-21 (MUC21) expression on the susceptibility to apoptosis. MUC21 transfection into HEK293 cells decreased the number of apoptotic cells in culture media containing etoposide or after ultraviolet light irradiation. We used Chinese hamster ovary (CHO) cell variants to investigate the importance of MUC21 glycosylation in the resistance to apoptosis. When MUC21 was expressed in CHO-K1 cells, it was glycosylated with sialyl T-antigen and the cells showed resistance to etoposide-induced apoptosis. MUC21 transfection into Lec2 cells, a variant of CHO cells lacking sialylation of glycans, revealed that the presence of nonsialylated T-antigen also renders cells resistant to etoposide-induced apoptosis. MUC21 was transfected into ldlD cells and the glycosylation was manipulated by supplementation to the medium. Nonsupplemented cells and cells supplemented with N-acetylgalactosamine showed no resistance to etoposide-induced apoptosis. In contrast, these cells supplemented with N-acetylgalactosamine plus galactose expressed sialyl T-antigen and exhibited resistance to etoposide-induced apoptosis. Finally, galectin-3 knockdown in MUC21 transfectants of HEK293 cells did not significantly affect MUC21-dependent induction of apoptosis resistance. The results suggest that T-antigen with or without sialic acid is essential to the antiapoptotic effect of MUC21., (© 2022. The Author(s).)

Background: KRAS is one of the most frequently mutated oncogenes in various cancers, and several novel KRAS G12C direct inhibitors are now in clinical trials. Here, we characterised the anti-tumour efficacy of ASP2453, a novel KRAS G12C inhibitor, in preclinical models of KRAS G12C-mutated cancer., Methods: We evaluated the in vitro and in vivo activity of ASP2453, alone or in combination with targeted agents and immune checkpoint inhibitors, in KRAS G12C-mutated cancer cells and xenograft models. We also assessed pharmacological differences between ASP2453 and AMG 510, another KRAS G12C inhibitor, using an SPR assay, washout experiments and an AMG 510-resistant xenograft model., Results: ASP2453 potently and selectively inhibited KRAS G12C-mediated growth, KRAS activation and downstream signalling in vitro and in vivo, and improved the anti-tumour effects of targeted agents and immune checkpoint inhibitors. Further, ASP2453 had more rapid binding kinetics to KRAS G12C protein and showed more potent inhibitory effects on KRAS activation and cell proliferation after washout than AMG 510. ASP2453 also induced tumour regression in an AMG 510-resistant xenograft model., Conclusions: ASP2453 is a potential therapeutic agent for KRAS G12C-mutated cancer. ASP2453 showed efficacy in AMG 510-resistant tumours, even among compounds with the same mode of action., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The symbiotic nitrogen-fixing bacterium Bradyrhizobium japonicum is critical to the agro-industrial production of soybean because it enables the production of high yields of soybeans with little use of nitrogenous fertilizers. The FixL and FixJ two-component system (TCS) of this bacterium ensures that nitrogen fixation is only stimulated under conditions of low oxygen. When it is not bound to oxygen, the histidine kinase FixL undergoes autophosphorylation and transfers phosphate from adenosine triphosphate (ATP) to the response regulator FixJ, which, in turn, stimulates the expression of genes required for nitrogen fixation. We purified full-length B. japonicum FixL and FixJ proteins and defined their structures individually and in complex using small-angle x-ray scattering, crystallographic, and in silico modeling techniques. Comparison of active and inactive forms of FixL suggests that intramolecular signal transduction is driven by local changes in the sensor domain and in the coiled-coil region connecting the sensor and histidine kinase domains. We also found that FixJ exhibits conformational plasticity not only in the monomeric state but also in tetrameric complexes with FixL during phosphotransfer. This structural characterization of a complete TCS contributes both a mechanistic and evolutionary understanding to TCS signal relay, specifically in the context of the control of nitrogen fixation in root nodules., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background/aim: Maternal embryonic leucine zipper kinase (MELK) is categorized as a member of AMP-activated protein kinase families. Various MELK-associated cellular and biological processes affect multiple stages of tumorigenesis. The aim of the present study was to clarify the relationship between MELK expression and hepatocellular carcinoma (HCC) clinicopathological features., Materials and Methods: In thirty conserved frozen primary HCC and non-HCC samples MELK mRNA expression was examined by quantitative real-time polymerase chain reaction (PCR)., Results: HCC tissues exhibited significantly higher expression levels compared to non-cancerous tissues. MELK expression had a statistically parallel correlation between tumor diameter and protein induced by vitamin K absence or antagonist II (PIVKA-II). The overall survival (OS) and recurrence-free survival (RFS) of the low MELK mRNA expression group was significantly longer than that of the high MELK mRNA expression group., Conclusion: MELK expression in HCC is extremely intense compared to its expression reported in other types of cancer. MELK could be a promising effective tumor marker of HCC and further consideration is needed., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Therapeutic effectiveness against metastatic or even locally advanced pancreatic ductal adenocarcinoma (PDAC) is dismal, with 5-year survival less than 5%. Even in patients who undergo potentially curative resection, most patients' tumors recur in the liver. Improving therapies targeting or preventing liver metastases is crucial for improving prognosis. To identify genes suppressing metastasis, a genome-wide shRNA screen was done using the human non-metastatic PDAC cell line, S2-028. After identification of candidates, functional validation was done using intrasplenic and orthotopic injections in athymic mice. HMP19 strongly inhibited metastasis but also partially attenuated tumor growth in the pancreas. Knockdown of HMP19 increased localization of activated ERK1/2 in the nucleus, corresponding to facilitated cell proliferation, decreased p27(Kip1) and increased cyclin E1. Over-expression of HMP19 exerted the opposite effects. Using a tissue microarray of 84 human PDAC, patients with low expression of HMP19 showed significantly higher incidence of liver metastasis (p = 0.0175) and worse prognosis (p = 0.018) after surgery. HMP19, a new metastasis/tumor suppressor in PDAC, appears to alter signaling that leads to cell proliferation and appears to offer prognostic value in human PDAC., (© 2016 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of Union for International Cancer Control.)

Background: The incidence of pathological lymph node metastases in patients with gastric cancer is 5% to 10%, which means that approximately 90% of patients with gastric cancer may undergo unnecessary lymphadenectomy. The precise intraoperative diagnosis of sentinel lymph node (SN) metastases is essential. The purpose of the current study was to verify the usefulness of a rapid reverse transcriptase-polymerase chain reaction (RT-PCR) system compared with hematoxylin and eosin staining for such diagnoses., Methods: A total of 113 patients with clinical T1-T2 (cT1-T2) gastric cancer, including 73 patients with cT1cN0 disease with a tumor diameter <4 cm, were enrolled in the current study. SNs were identified by a radioisotope method. Carcinoembryonic antigen and cytokeratin 19 were used as markers for RT-PCR and the cutoff values were set using 1701 lymph nodes harvested from 157 patients with gastric cancer., Results: SNs were detected in all 113 patients. Sensitivity and accuracy for detection by paraffin section were both 100% in patients with cT1 disease and were 60% and 90%, respectively, in patients with cT2 disease. The sensitivity of RT-PCR for the detection of pathological SN metastases was 92.3%. Furthermore, 11 patients had SN metastases detected only by RT-PCR, and these patients had frequent lymphatic invasion. Hematoxylin and eosin staining detected SN metastases in 6 of 73 patients with cT1cN0 gastric cancer; RT-PCR and frozen section detected SN metastases in 6 and 4 of these patients, respectively. Accordingly, the sensitivity of RT-PCR and frozen section for the detection of those pathological SN metastases were 100% and 66.6%, respectively., Conclusions: The rapid RT-PCR system appears to have clinical usefulness for the intraoperative detection of SN metastases in patients with gastric cancer., (© 2015 American Cancer Society.)

Background: Esophageal squamous cell carcinoma is an aggressive gastrointestinal tract cancer. To date, the presence of circulating tumor cells (CTC) has been reported as a prognostic factor in peripheral blood from patients with gastrointestinal cancers., Methods: The CellSearch system was used to isolate and enumerate CTCs. A total of 90 patients with esophageal squamous cell carcinoma who received chemotherapy or chemoradiotherapy were enrolled. Peripheral blood specimens were collected before and after treatments., Results: At baseline analysis, CTCs were detected in 25 patients (27.8 %). Overall survival was significantly shorter in patients with than without CTCs. Follow-up blood specimens were obtained from 71 patients. Partial response, stable disease, and progressive disease after treatment were seen in 32, 12, and 27 patients, respectively. CTC positivity after treatment in the progressive disease group (40.7 %) was significantly higher than that of the partial response group (6.3 %). Patients with a change in CTC status from positive to negative had a good prognosis as well as patients without baseline CTCs., Conclusions: Evaluation of CTCs may be a promising indicator for predicting tumor prognosis and the clinical efficacy of chemotherapy or chemoradiation therapy in patients with esophageal squamous cell carcinoma.

Background: The transcription factor nuclear factor (erythroid-2)-related factor 2 (Nrf2) was originally identified as a critical regulator of intracellular anti-oxidants and of phase II detoxification enzymes through its transcriptional up-regulation of many anti-oxidant response element (ARE)-containing genes. Nrf2 protects not only normal cells but also cancer cells from cellular stress, and enhances cancer cell survival. Some studies have shown that Nrf2 expression in cancer patients has clinical significance. However, there has been no comprehensive analysis of the nuclear expression level of Nrf2 in gastrointestinal cancer cells. In this study we aimed to immunohistochemically evaluate the expression of Nrf2, and to assess its clinical significance in gastric cancer., Methods: A total of 175 gastric cancer patients who received R0 gastrectomy with standard lymph node dissection were enrolled. We immunohistochemically evaluated Nrf2 expression in the paraffin-embedded surgically resected specimens of these 175 patients. Group differences were analyzed using the χ (2) test, Fisher's exact test, and the Mann-Whitney U test. Associations between Nrf2 expression and clinicopathological features, including clinical outcome, were assessed using univariate and multivariate analyses, and Kaplan-Meier curves with the log-rank test, respectively., Results: Nrf2 immunoreactivity was predominantly identified in the nucleus of gastric cancer cells. Nrf2 positivity was closely associated with tumor size, tumor depth, lymph node metastases, lymphovascular invasion, histology and stage (p < 0.05 for all). A log-rank test indicated that the overall survival of the Nrf2-positive group was significantly poorer than that of the Nrf2-negative group (p < 0.01). And, positive Nrf2 expression was significantly associated with resistance to 5FU-based adjuvant chemotherapy (p = 0.024)., Conclusions: Nrf2 expression was positively associated with aggressive tumor behavior in gastric cancer. This result suggests that Nrf2 expression in gastric cancer is a potential indicator of worse prognosis.

Cyclin-dependent kinase subunit 2 (CKS2) is a cyclin-dependent kinase subunit (CKS) family member that participates in cell cycle regulation. Few studies have investigated its involvement in esophageal squamous cell carcinoma (ESCC). The aim of the present study was to assess the clinical significance of CKS2 in ESCC. We used immunohistochemistry to study the clinicopathologic significance of CKS2 protein expression in 121 patients with ESCC. Using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), we examined the expression of CKS2 mRNA in tumors and the corresponding normal esophageal tissues that were obtained from 62 patients. Finally, siRNA-mediated attenuation of CKS2 expression was examined in vitro. CKS2 protein expression was significantly correlated with depth of tumor invasion, clinical stage, lymphatic invasion and distant metastasis (p=0.033, 0.028, 0.041 and 0.009, respectively). CKS2 mRNA expression was higher in cancer tissue than in corresponding normal tissue (p<0.001). Patients with positive-CKS2 protein expression had a poorer five year survival frequency than patients who did not express CKS2 protein (p=0.025). In vitro, siRNA-mediated suppression of CKS2 slowed the growth rate of ESCC cells compared to control cells (p<0.001). The evaluation of CKS2 expression is useful for predicting the cause of malignant tumors and the prognosis of patients with ESSC.

Background: Human leukocyte antigen (HLA)-F is a nonclassical HLA class I molecule that shows aberrant expression in cancer cells. Although the clinical implications of HLA-F expression in cancer patients have been described, the specific significance of this antigen in gastrointestinal cancer remains unclear. The present study examined the expression pattern and clinical implications of HLA-F in gastric adenocarcinoma., Patients and Methods: HLA-F expression was assessed in 179 patients by immunohistochemistry, and its association with clinical parameters including patient survival was analyzed., Results: HLA-F expression was positive in 30.7% (55/179) of patients and in 50.0% (90/179) of peritumoral infiltrating lymphocytes. HLA-F expression in gastric adenocarcinoma was significantly correlated with the depth of invasion, nodal involvement, and lymphatic and venous invasions (P < 0.01, all). HLA-F positivity of infiltrating cells near the tumor showed no correlation with clinicopathological features. HLA-F-positive patients had a significantly worse prognosis than HLA-F-negative patients (P = 0.012). However, HLA-F expression was not an independent prognostic factor in multivariate analysis., Conclusions: The present study provides the first evidence that neo-HLA-F expression is of clinical significance in gastric adenocarcinoma. HLA-F expression in gastric adenocarcinoma may promote the aggressive behavior of tumors by suppressing the activity of antitumor immune effector cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Delta-like ligand 4 (DLL4)-Notch signaling plays a key role in tumor neovascular development and angiogenesis during tumor growth. The clinical significance of DLL4 expression in gastric cancer has not been clarified., Methods: Gastric cancer cell lines and 180 gastric cancer patients were enrolled. DLL4 expression in gastric cancer cells and stroma was identified and evaluated immunohistochemically. The association between DLL4 and clinicopathological factors was also assessed., Results: DLL4 expression was identified in the cellular membrane and cytoplasm of gastric cancer cells by immunoblotting and immunohistochemical staining. DLL4 positivity in cancer cells and stroma was found in 88 (48%) and 41 (22%) of the 180 gastric cancer patients respectively. Both cancer and stromal DLL4 expression significantly correlated with more advanced tumor depth, nodal involvement, and lymphatic and venous invasion. A strongly positive association between cancerous and stromal DLL4 expression was identified (p < 0.01). Both cancerous and stromal DLL4 expression were prognostic markers in gastric cancer as determined by univariate analysis., Conclusions: Cancerous and stromal DLL4 expression was found in 48% and 22% in gastric cancer, and significantly affected postoperative clinical outcomes. Cancerous and stromal DLL4 expression may be an effective target of anti-DLL4 treatment in gastric cancer.

Background: Since antitumor immune reactions between tumors and intratumoral immunocytes have been verified in several human tumors, immunological therapeutic strategies must be considered to obtain the proper efficacy of tumor shrinkage under these conditions. Human leukocyte antigen (HLA) class I expression in cancer cells and degree of infiltration of regulatory T cells (Tregs) in the stroma have been regarded as important markers of antitumor immune reactions in the context of independent immunological mechanisms. In the current study, we investigated HLA class I expression and Treg cells infiltration in gastric cancer and discussed the clinical implications of this combinatory analysis in gastric cancer., Patients and Methods: A total of 141 gastric cancer patients who received R0 gastrectomy at Kagoshima University Hospital were studied. Immunohistochemically, in 141 gastric cancer patients, HLA class I expression and Treg cell infiltration in cancerous tissue were evaluated using HLA class I (EMR8-5) and forkhead box p3 (FOXP3) monoclonal antibodies. The correlation between clinical factors and tumor-infiltrating Treg cells was analyzed., Results: HLA class I expression was positively associated with depth of tumor invasion (P < 0.05). Infiltration of Foxp3-positive cells did not correlate with any clinicopathological markers. HLA class I expression had no association with Treg cell infiltration (r = 0.04). A better postoperative outcome was associated with fewer numbers of Treg infiltration (P = 0.034). A combination of HLA and Treg analysis may lead to a more accurate prediction of postoperative outcome (P = 0.02)., Conclusions: Two different antitumor immunological markers, Treg infiltration and HLA class I expression, affected clinicopathological factors in gastric cancer by different mechanisms. Thus, an immunological combination of HLA class I expression and Treg cell infiltration may more accurately predict postoperative outcome. Immunological balance needs to be restored after evaluation of each immunological deficit in gastric cancer.

Background: Interactions of stromal hyaluronic acid (HA) with its binding protein RHAMM (receptor for HA-mediated motility) (CD168) have been reported to affect tumor extension and the migration of crucial molecules to promote tumor progression and metastases. Cancerous CD168 expression is correlated with aggressive biological features in several cancers. However, the clinical implications of CD168 positivity in gastric cancer have remained unclear., Methods: We examined the CD168 expression of 196 consecutive gastric cancer patients by immunohistochemistry. According to CD168 positivity, the 196 gastric cancer patients were divided into two groups (57 CD168-positive and 139 CD168-negative patients). The correlation between CD168 expression and clinicopathological factors (age, sex, histology, tumor depth, lymph node status, and vessel invasion) was evaluated according to the Japanese Classification of Gastric Carcinoma., Results: Cancerous CD168 expression was detectable in 57 of the 196 tumors (29%). CD168 positivity was significantly correlated with the depth of invasion, nodal involvement, and vessel invasion (p < 0.01). Survival analysis of the 196 gastric cancer patients showed that the CD168-positive group had a significantly higher mortality than the CD168-negative group (p < 0.01). In terms of a correlation with CD168 positivity at separate clinical stages, a significance difference was only found in stages II and III. Multivariate analysis revealed that CD168 expression was a significant independent prognostic marker (p = 0.013) after depth of invasion (p < 0.005) and nodal involvement (p < 0.01)., Conclusion: Our results suggest that cancerous CD168 positivity is strongly related to the invasion and metastasis of gastric cancer tumors. These results suggest that cancerous CD168 expression can be used as a prognostic marker of gastric cancer owing to its interactions with stromal hyaluronic acid.

Background: CD166 is one of the cell-surface immunoglobulins, and is well known to regulate leukocyte mobility. Its expression is associated with aggressive tumor behavior. CD166 expression is a prognostic marker in several cancers, but the predictive value of CD166 expression in gastric cancer has not been clarified yet., Patients and Methods: A total of 142 gastric cancer patients who consecutively received curative gastrectomy in Kagoshima University Hospital were enrolled in the current study. The patients were composed of 99 men and 43 women, ranging in age from 42 to 84 years (mean 63 years). Cancerous CD166 expression was evaluated immunohistochemically., Results: Cancerous CD166 expression was identified in not only cellular membrane but also cytoplasm. The rates of membranous and cytoplasmic CD166 positivities were 25.4% and 34.4%, respectively. Cytoplasmic and membranous CD166 positivities were significantly correlated with nodal involvement and vascular invasion. Survival analysis of the 142 gastric cancer patients revealed that membranous CD166-positive group (median survival 18.6 months, range 0.3-104.5 months) had a significantly poorer outcome than CD166-negative group (median 25.7 months range 1.4-106 months) (P < 0.05)., Conclusions: Membranous CD166-positivity may contribute to one of the promising prognostic markers in gastric cancer.

The effects of hyaluronic acid, which comprises the cumulus intercellular matrix, on Ca(2+) influx, lactate dehydrogenase (LDH) activity, and cyclic AMP synthesis in canine sperm during capacitation was investigated. Ejaculated sperm were collected from 10 Beagle dogs and the sperm were incubated for 4 hr in Eagle's MEM containing 10 microg/ml of hyaluronic acid. The percentages of actively motile sperm, hyperactivated sperm (HA-sperm), acrosome-reacted sperm (AR-sperm), and sperm labeled with fluoresceinated Ca(2+) indicator (Ca(2+)-labeled sperm) were evaluated to assess Ca(2+) influx into the sperm. LDH activity and cAMP concentration were measured in homogenized sperm. The mean percentages of motile sperm, HA-sperm, and Ca(2+)-labeled sperm in the MEM containing hyaluronic acid were higher than in the control medium (P<0.05, 0.05, and 0.01, respectively), but there was no difference between the percentages of AR-sperm. Mean LDH activity and mean cAMP concentration were also significantly higher than the control values (P<0.05). The percentages of HA-sperm correlated with those of Ca(2+)-labeled sperm (r(2)=0.810). The results indicate that hyaluronic acid increases Ca(2+) influx, LDH activity, and cAMP synthesis in canine ejaculated sperm during capacitation in vitro.

We measured changes in membrane potential (MP), extra- and intracellular potassium and intracellular sodium ion activities (aKo, aKi, and aNai) in papillary muscle of the guinea-pig heart induced by repetitive stimulation at various frequencies and periods in vitro, using double-barrelled ion-selective microelectrodes. Stimulation (2-4 Hz) depolarized the cells, and termination of stimulation induced hyperpolarization. The aKi and aNai (stimulated at 0.2 Hz) were 92.3 +/- 4.6 and 7.8 +/- 2.0 mM (mean +/- S.D.), respectively. Prolongation of the stimulation period (0.5-2 min) increased aNai time-dependently, but there was no further increase by stimulation for longer than 3 min. The increase in aNai was dependent on stimulus frequency. After termination of the stimulation aiNa declined exponentially. aKi slightly decreased by the stimulation (3-4 Hz). aKo increased during the stimulation period, and decreased below the initial level after termination of the stimulation. Results suggest that in guinea-pig papillary muscle, the contribution of an electrogenic component of Na-K pump to the hyperpolarization after stimulation would be small, since the hyperpolarization could be explained mostly by depletion of aKo induced by Na-K pump activity.

We have developed a new model for electrophysiological studies on ischemic myocardium in situ. Pieces of ventricular tissue (1 X 1 mm) excised from the neonatal rat heart were transplanted into the renal capsular space of the adult rat of the pure strain F 344-NSlc. Electrophysiological properties of the transplanted cells during normal circulation and during ischemia induced by ligation of the renal artery were examined in situ 7-10 days later, when the tissues were fully vascularized, by means of conventional microelectrode techniques. Transmembrane potentials varied in individual cells, ranging from -60 to -90 mV. The amplitude, duration, and configuration of spontaneously occurring action potentials recorded from the transplanted cells were similar to those obtained in neonatal and adult rat myocardium. At 2-5 min after ligation of the renal artery, the frequency of the action potentials decreased markedly and their duration was transiently prolonged and then shortened. During the initial stage of ischemia (5-15 min), most of the cells were markedly depolarized and spontaneous activity ceased. Within 20-30 min, after the ligation, the cells did not respond to electrical stimulation. Reperfusion of the tissue by releasing the ligation restored the electrical activity of the cells within 15 min when a certain limited time of ischemia (30-40 min) was not exceeded.

Two cases of hepatocellular carcinoma (HCC) were described, in whom hypercholinesterasemia was found. Histochemical examinations revealed that there was a significantly increase in enzyme activity of cholinesterase in liver tissue slice obtained from the part of carcinoma in case 1. It was found that cholinesterase activity in homogenized liver tissue in part of carcinoma was much higher than that of non-carcinoma, and even in other HCC cases, hepatic cirrhosis and control liver tissues. These results suggested that HCC cells were capable of producing cholinesterase and, therefore, that hypercholinesterasemia was an additional paraneoplastic syndrome in HCC.