The analysis on groundwater storage variations from GRACE/GRACE-FO in recent 20 years driven by influencing factors and prediction in Shandong Province, China.

Monitoring and predicting the regional groundwater storage (GWS) fluctuation is an essential support for effectively managing water resources. Therefore, taking Shandong Province as an example, the data from Gravity Recovery and Climate Experiment (GRACE) and GRACE Follow-On (GRACE-FO) is used to invert GWS fluctuation from January 2003 to December 2022 together with Watergap Global Hydrological Model (WGHM), in-situ groundwater volume and level data. The spatio-temporal characteristics are decomposed using Independent Components Analysis (ICA), and the impact factors, such as precipitation and human activities, which are also analyzed. To predict the short-time changes of GWS, the Support Vector Machines (SVM) is adopted together with three commonly used methods Long Short-Term Memory (LSTM), Singular Spectrum Analysis (SSA), Auto-Regressive Moving Average Model (ARMA), as the comparison. The results show that: (1) The loss intensity of western GWS is significantly greater than those in coastal areas. From 2003 to 2006, GWS increased sharply; during 2007 to 2014, there exists a loss rate - 5.80 ± 2.28 mm/a of GWS; the linear trend of GWS change is - 5.39 ± 3.65 mm/a from 2015 to 2022, may be mainly due to the effect of South-to-North Water Diversion Project. The correlation coefficient between GRACE and WGHM is 0.67, which is consistent with in-situ groundwater volume and level. (2) The GWS has higher positive correlation with monthly Global Precipitation Climatology Project (GPCP) considering time delay after moving average, which has the similar energy spectrum depending on Continuous Wavelet Transform (CWT) method. In addition, the influencing facotrs on annual GWS fluctuation are analyzed, the correlation coefficient between GWS and in-situ data including the consumption of groundwater mining, farmland irrigation is 0.80, 0.71, respectively. (3) For the GWS prediction, SVM method is adopted to analyze, three training samples with 180, 204 and 228 months are established with the goodness-of-fit all higher than 0.97. The correlation coefficients are 0.56, 0.75, 0.68; RMSE is 5.26, 4.42, 5.65 mm; NSE is 0.28, 0.43, 0.36, respectively. The performance of SVM model is better than the other methods for the short-term prediction.

A PANoptosis pattern to predict prognosis and immunotherapy response in head and neck squamous cell carcinoma.

Individuals diagnosed with head and neck squamous cell carcinoma (HNSCC) experience a significant occurrence rate and are susceptible to premature spreading, resulting in a bleak outlook. Therapeutic approaches, such as chemotherapy, targeted therapy, and immunotherapy, may exhibit primary and acquired resistance during the advanced phases of HNSCC. There is currently no viable solution to tackle this issue. PANoptosis-a type of non-apoptotic cell death-is a recently identified mechanism of cellular demise that entails communication and synchronization among thermal apoptosis, apoptosis, and necrosis mechanisms. However, the extent to which PANoptosis-associated genes (PRG) contribute to the forecast and immune reaction of HNSCC remains mostly undisclosed. The present study aimed to thoroughly analyze the potential importance of PRG in HNSCC and report our discoveries. We systematically analyzed 19 PRG from previous studies and clinical data from HNSCC patients to establish a PAN-related signature and assess its prognostic, predictive potential. Afterward, the patient information was separated into two gene patterns that corresponded to each other, and the analysis focused on the connection between patient prognosis, immune status, and cancer immunotherapy. The PAN score was found to correlate with survival rates, immune systems, and cancer-related pathways. We then validated the malignant role of CD27 among them in HNSCC. In summary, we demonstrated the effectiveness of PAN.Score-based molecular clustering and prognostic features in predicting the outcome of HNSCC. The discovery we made could enhance our comprehension of the significance of PAN.Score in HNSCC and facilitate the development of more effective treatment approaches.

Nuclear export of circular RNA.

Circular RNAs (circRNAs), which are increasingly being implicated in a variety of functions in normal and cancerous cells1-5, are formed by back-splicing of precursor mRNAs in the nucleus6-10. circRNAs are predominantly localized in the cytoplasm, indicating that they must be exported from the nucleus. Here we identify a pathway that is specific for the nuclear export of circular RNA. This pathway requires Ran-GTP, exportin-2 and IGF2BP1. Enhancing the nuclear Ran-GTP gradient by depletion or chemical inhibition of the major protein exporter CRM1 selectively increases the nuclear export of circRNAs, while reducing the nuclear Ran-GTP gradient selectively blocks circRNA export. Depletion or knockout of exportin-2 specifically inhibits nuclear export of circRNA. Analysis of nuclear circRNA-binding proteins reveals that interaction between IGF2BP1 and circRNA is enhanced by Ran-GTP. The formation of circRNA export complexes in the nucleus is promoted by Ran-GTP through its interactions with exportin-2, circRNA and IGF2BP1. Our findings demonstrate that adaptors such as IGF2BP1 that bind directly to circular RNAs recruit Ran-GTP and exportin-2 to export circRNAs in a mechanism that is analogous to protein export, rather than mRNA export.

Structural basis of nucleosomal H4K20 recognition and methylation by SUV420H1 methyltransferase.

Histone lysine methyltransferase SUV420H1, which is responsible for site-specific di-/tri-methylation of histone H4 lysine 20 (H4K20), has crucial roles in DNA-templated processes, including DNA replication, DNA damage repair, and chromatin compaction. Its mutations frequently occur in human cancers. Nucleosomes containing the histone variant H2A.Z enhance the catalytic activities of SUV420H1 on H4K20 di-methylation deposition, regulating early replication origins. However, the molecular mechanism by which SUV420H1 specifically recognizes and deposits H4K20 methyl marks on nucleosomes remains poorly understood. Here we report the cryo-electron microscopy structures of SUV420H1 associated with H2A-containing nucleosome core particles (NCPs), and H2A.Z-containing NCPs. We find that SUV420H1 makes extensive site-specific contacts with histone and DNA regions. SUV420H1 C-terminal domain recognizes the H2A-H2B acidic patch of NCPs through its two arginine anchors, thus enabling H4K20 insertion for catalysis specifically. We also identify important residues increasing the catalytic activities of SUV420H1 bound to H2A.Z NCPs. In vitro and in vivo functional analyses reveal that multiple disease-associated mutations at the interfaces are essential for its catalytic activity and chromatin state regulation. Together, our study provides molecular insights into the nucleosome-based recognition and methylation mechanisms of SUV420H1, and a structural basis for understanding SUV420H1-related human disease.

Urinary microRNAs in sepsis function as a novel prognostic marker.

Renal dysfunction is a common complication of sepsis. Early diagnosis and prompt treatment of sepsis with renal insufficiency are crucial for improving patient outcomes. Diagnostic markers can help identify patients at risk for sepsis and AKI, allowing for early intervention and potentially preventing the development of severe complications. The aim of the present study was to investigate the expression difference of urinary microRNAs (miRNAs/miRs) in elderly patients with sepsis and secondary renal insufficiency, and to evaluate their diagnostic value in these patients. In the present study, RNA was extracted from urine samples of elderly sepsis-related acute renal damage patients and the expression profiles of several miRNAs were analyzed. In order to evaluate the expression profile of several miRNAs, urine samples from elderly patients with acute renal damage brought on by sepsis were obtained. RNA extraction and sequencing were then performed on the samples. Furthermore, multiple bioinformatics methods were used to analyze miRNA profiles, including differential expression analysis, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of different miRNA target genes, to further explore miRNAs that are suitable for utilization as biomarkers. A total of four miRNAs, including hsa-miR-31-5p, hsa-miR-151a-3p, hsa-miR-142-5p and hsa-miR-16-5p, were identified as potential biological markers and were further confirmed in sepsis using reverse transcription-quantitative PCR. The results of the present study demonstrated that the four urinary miRNAs were differentially expressed and may serve as specific markers for prediction of secondary acute kidney injury in elderly patients with sepsis.

Synthesis and identification of a novel skeleton of N-(pyridin-3-yl) proline as a selective CDK4/6 inhibitor with anti-breast cancer activities.

CDK4/6 were attractive chemotherapeutic targets for the treatment of malignant tumors, CDK4/6 selective inhibitors have made outstanding contributions in the treatment of breast cancer. However, these inhibitors share a single skeleton of N-(pyridin-2-yl) pyrimidin-2-amine which cannot overcome the side effects in clinical application. In our previous study, an N'- acetylpyrrolidine-1-carbohydrazide was hit as the initial fragment by analyzing the active site characteristics of CDK6. Two series of N-(pyridin-3-yl) proline were obtained by fragment growth method. The QSAR study was carried out according to the in vitro activities data against CDK4/6, and two compounds 7c and 7p with potent inhibitory activities were found to interact with CDK4 in different binding conformation. They showed potential inhibition of cell proliferation against the breast cancer cell, and 7c exhibited promised anti-breast cancer effect in vivo.

Regional Crustal Vertical Deformation Driven by Terrestrial Water Load Depending on CORS Network and Environmental Loading Data: A Case Study of Southeast Zhejiang.

Monitoring regional terrestrial water load deformation is of great significance to the dynamic maintenance and hydrodynamic study of the regional benchmark framework. In view of the lack of a spatial interpolation method based on the GNSS (Global Navigation Satellite System) elevation time series for obtaining terrestrial water load deformation information, this paper proposes to employ a CORS (Continuously Operating Reference Stations) network combined with environmental loading data, such as ECMWF (European Centre for Medium-Range Weather Forecasts) atmospheric data, the GLDAS (Global Land Data Assimilation System) hydrological model, and MSLA (Mean Sea Level Anomaly) data. Based on the load deformation theory and spherical harmonic analysis method, we took 38 CORS stations in southeast Zhejiang province as an example and comprehensively determined the vertical deformation of the crust as caused by regional terrestrial water load changes from January 2015 to December 2017, and then compared these data with the GRACE (Gravity Recovery and Climate Experiment) satellite. The results show that the vertical deformation value of the terrestrial water load in southeast Zhejiang, as monitored by the CORS network, can reach a centimeter, and the amplitude changes from -1.8 cm to 2.4 cm. The seasonal change is obvious, and the spatial distribution takes a ladder form from inland to coastal regions. The surface vertical deformation caused by groundwater load changes in the east-west-south-north-central sub-regions show obvious fluctuations from 2015 to 2017, and the trends of the five sub-regions are consistent. The amplitude of surface vertical deformation caused by groundwater load change in the west is higher than that in the east. We tested the use of GRACE for the verification of CORS network monitoring results and found a relatively consistent temporal distribution between both data sets after phase delay correction on GRACE, except for in three months-November in 2015, and January and February in 2016. The results show that the comprehensive solution based on the CORS network can effectively improve the monitoring of crustal vertical deformation during regional terrestrial water load change.

Structural insights into proteolytic activation of the human Dispatched1 transporter for Hedgehog morphogen release.

The membrane protein Dispatched (Disp), which belongs to the RND family of small molecule transporters, is essential for Hedgehog (Hh) signaling, by catalyzing the extracellular release of palmitate- and cholesterol-modified Hh ligands from producing cells. Disp function requires Furin-mediated proteolytic cleavage of its extracellular domain, but how this activates Disp remains obscure. Here, we employ cryo-electron microscopy to determine atomic structures of human Disp1 (hDisp1), before and after cleavage, and in complex with lipid-modified Sonic hedgehog (Shh) ligand. These structures, together with biochemical data, reveal that proteolytic cleavage opens the extracellular domain of hDisp1, removing steric hindrance to Shh binding. Structure-guided functional experiments demonstrate the role of hDisp1-Shh interactions in ligand release. Our results clarify the mechanisms of hDisp1 activation and Shh morphogen release, and highlight how a unique proteolytic cleavage event enabled acquisition of a protein substrate by a member of a family of small molecule transporters.

Downregulation of LINC01296 suppresses non-small-cell lung cancer via targeting miR-143-3p/ATG2B.

The 5-year survival rate of lung cancer is one of the lowest among various malignant tumors. Long noncoding RNAs (lncRNAs), noncoding RNAs longer than 200 nucleotides, can function either as tumor suppressors or as oncogenes. The aim of this study is to investigate the function of lncRNA LINC01296 and its molecular mechanism in non-small-cell lung cancer (NSCLC). According to the Gene Expression Omnibus database, 10 differentially expressed lncRNAs in NSCLC cells and patient tissues are upregulated. LINC01296 is the one with the most significant overexpression. Knockdown of LINC01296 inhibits the growth and migration, arrests the cell cycle, and promotes the apoptosis of NSCLC cells. Knocking down LINC01296 in vivo suppresses tumor growth and metastasis. LINC01296 also acts as the sponge of miR-143-3p. Lowering the expression of LINC01296 leads to decreased expression of autophagy-related 2B (ATG2B), a target gene of miR-143-3p. Moreover, downregulation of LINC01296 promotes paclitaxel sensitivity in NSCLC. These results demonstrated that the LINC01296/miR-143-3p/ATG2B axis is crucial in promoting the development of NSCLC and paclitaxel resistance. Our study may provide new ideas for the further research of clinical chemotherapy of NSCLC in the near future.

Modulation of the Wound Healing through Noncoding RNA Interplay and GSK-3ß/NF-κB Signaling Interaction.

Diabetic foot ulcers are seriously endangering the physical and mental health of patients. Due to the long duration of inflammation, the treatment of nonhealing wounds in diabetes is one of the most prominent healthcare problems in the world. The nuclear factor kappa B (NF-κB) signaling pathway, a classical pathway that triggers inflammatory response, is regulated by many regulators, such as glycogen synthase kinase 3 beta (GSK-3ß). Noncoding RNAs, a large class of molecules that regulate gene expression at the posttranscriptional or posttranslational level, play an important role in various stages of wound healing, especially in the stage of inflammation. Herein, we summarized the roles of noncoding RNAs in the NF-κB/GSK-3ß signaling, which might provide new ideas for the treatment of diabetic wound healing.

MicroRNA-486-5p Suppresses Lung Cancer via Downregulating mTOR Signaling In Vitro and In Vivo.

Lung cancer is one of the central causes of tumor-related deaths globally, of which non-small cell lung cancer (NSCLC) takes up about 85%. As key regulators of various biological processes, microRNAs (miRNAs) have been verified as crucial factors in NSCLC. To elucidate the role of miR-486-5p in the mTOR pathway, we investigated its role in NSCLC and related signaling. Our results confirmed that miR-486-5p was downregulated in most of human NSCLC tissue samples and cell lines. Further study confirmed that it inhibited NSCLC through repression of the mTOR pathway via targeting both ribosomal proteins S6 kinase A1 (RPS6KA1, RSK) and ribosomal proteins S6 kinase B1 (RPS6KB1, p70S6K), which are critical components of the mTOR signaling. Additionally, miR-486-5p impeded tumor growth in vivo and inhibited tumor metastasis through repression of the epithelial-mesenchymal transition (EMT). Taken together, our study verified the role that miR-486-5p exerts in NSCLC, and its expression pattern in the different stages and morphologies of NSCLC makes it a promising biomarker in the early diagnosis of the disease.

Molecular basis of nucleosomal H3K36 methylation by NSD methyltransferases.

Histone methyltransferases of the nuclear receptor-binding SET domain protein (NSD) family, including NSD1, NSD2 and NSD3, have crucial roles in chromatin regulation and are implicated in oncogenesis1,2. NSD enzymes exhibit an autoinhibitory state that is relieved by binding to nucleosomes, enabling dimethylation of histone H3 at Lys36 (H3K36)3-7. However, the molecular basis that underlies this mechanism is largely unknown. Here we solve the cryo-electron microscopy structures of NSD2 and NSD3 bound to mononucleosomes. We find that binding of NSD2 and NSD3 to mononucleosomes causes DNA near the linker region to unwrap, which facilitates insertion of the catalytic core between the histone octamer and the unwrapped segment of DNA. A network of DNA- and histone-specific contacts between NSD2 or NSD3 and the nucleosome precisely defines the position of the enzyme on the nucleosome, explaining the specificity of methylation to H3K36. Intermolecular contacts between NSD proteins and nucleosomes are altered by several recurrent cancer-associated mutations in NSD2 and NSD3. NSDs that contain these mutations are catalytically hyperactive in vitro and in cells, and their ectopic expression promotes the proliferation of cancer cells and the growth of xenograft tumours. Together, our research provides molecular insights into the nucleosome-based recognition and histone-modification mechanisms of NSD2 and NSD3, which could lead to strategies for therapeutic targeting of proteins of the NSD family.

Antitumor Activity of Lipid-DNA Aptamer Modified T Lymphocytes in Carcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with no current effective therapeutics. One of the main reasons for the low efficacy of PDAC immunotherapy is the limited CD8+ T cell infiltration, without neo antigen present in PDAC. Aptamers represent single-stranded oligonucleotides which bind to specific targets with high specificity. We developed DNA conjugates and prepared diacyl phospholipid-aptamer XQ-2d which has potential for the targeted therapy and diagnosis of PDAC. In this study, flow cytometry and fluorescence microscopy were employed to assess whether the Lipo-XQ-2d probe could anchor on activated T cells to constitute ligands specifically recognizing PDAC PL45 cells. Flow cytometry was employed to determine cytotoxicity in activated T cells. Results showed that the Lipo-XQ-2d probe could be inserted into T cells, and was specifically bound to both T cells and PL45 cells. In addition, the Lipo-XQ-2d probe redirected T cells to kill PL45 cells in vitro and was not toxic to cells. In conclusion, lipid-DNA-aptamer-modified T-lymphocytes might effectively kill PDAC in vitro, supporting the clinical application of T cell adoptive immunotherapy.

Higher expression of miR-150-5p promotes tumorigenesis by suppressing LKB1 in non-small cell lung cancer.

Lung cancer is one of the most malignant tumors that can form in the human. MicroRNAs (MiRNAs) play significant role in tumor progression. Human lung cancer tissues and cell lines were used to determine miR-150-5p respectively, and Liver Kinase B1 (LKB1) expression using quantitative real-time PCR (qRT-PCR). The data analysis website Kaplan-Meier Plotter (database obtained from The Cancer Genome Atlas) was used to perform a survival analysis with LKB1 levels. Using the appropriate assays, the function of miR-150-5p was also detected in cellular proliferation, migration and cell apoptosis as well as cell cycle. Results revealed that miR-150-5p was upregulated in non-small cell lung cancer (NSCLC) tissue and cell lines. In NSCLC, miR-150-5p promoted cellular proliferation and migration, but decreased cellular apoptosis. Conversely, miR-150-5p inhibition suppressed cellular growth. These results further revealed a network of cellular signaling for miR-150-5p to target LKB1. Ectopic expression of LKB1 can mitigate the tumor-promoting function of miR-150-5p. Collectively, these results indicated that miR-150-5p may promote lung cancer by inhibiting the suppressor gene LKB1 in NSCLC.

Loss of a single methylation in 23S rRNA delays 50S assembly at multiple late stages and impairs translation initiation and elongation.

Ribosome biogenesis is a complex process, and dozens of factors are required to facilitate and regulate the subunit assembly in bacteria. The 2'-O-methylation of U2552 in 23S rRNA by methyltransferase RrmJ is a crucial step in late-stage assembly of the 50S subunit. Its absence results in severe growth defect and marked accumulation of pre50S assembly intermediates. In the present work, we employed cryoelectron microscopy to characterize a set of late-stage pre50S particles isolated from an Escherichia coli ΔrrmJ strain. These assembly intermediates (solved at 3.2 to 3.8 Å resolution) define a collection of late-stage particles on a progressive assembly pathway. Apart from the absence of L16, L35, and L36, major structural differences between these intermediates and the mature 50S subunit are clustered near the peptidyl transferase center, such as H38, H68-71, and H89-93. In addition, the ribosomal A-loop of the mature 50S subunit from ΔrrmJ strain displays large local flexibility on nucleotides next to unmethylated U2552. Fast kinetics-based biochemical assays demonstrate that the ΔrrmJ 50S subunit is only 50% active and two times slower than the WT 50S subunit in rapid subunit association. While the ΔrrmJ 70S ribosomes show no defect in peptide bond formation, peptide release, and ribosome recycling, they translocate with 20% slower rate than the WT ribosomes in each round of elongation. These defects amplify during synthesis of the full-length proteins and cause overall defect in protein synthesis. In conclusion, our data reveal the molecular roles of U2552 methylation in both ribosome biogenesis and protein translation.

Synthesis and biological evaluation of novel (E)-N'-benzylidene hydrazides as novel c-Met inhibitors through fragment based virtual screening.

C-Met plays a crucial role in the development and progression of neoplastic disease. Type II c-Met inhibitors recognise the inactive DFG-out conformation of the kinase, result in better anti-tumour effects due to synergistic effect against the other kinases. According to our previous works, an (E)-N'-benzylidene group was selected as the initial fragment. Two series of (E)-N'-benzylidene hydrazides were designed by fragment growth method. The inhibitory activities were in vitro investigated against c-Met and VEGFR-2. Compound 10b exhibited the most potent inhibitory activity against the c-Met inhibitor (IC50 = 0.37 nM). Compound 11b exhibited multi-target c-Met kinase inhibitory activity as a potential type II c-Met inhibitor (IC50 = 3.41 nM against c-Met; 25.34 nM against VEGFR-2). The two compounds also demonstrate the feasibility of fragment-based virtual screening method for drug discovery.

Identification of novel CDK2 inhibitors by a multistage virtual screening method based on SVM, pharmacophore and docking model.

Cyclin-dependent kinase 2 (CDK2) is the family of Ser/Thr protein kinases that has emerged as a highly selective with low toxic cancer therapy target. A multistage virtual screening method combined by SVM, protein-ligand interaction fingerprints (PLIF) pharmacophore and docking was utilised for screening the CDK2 inhibitors. The evaluation of the validation set indicated that this method can be used to screen large chemical databases because it has a high hit-rate and enrichment factor (80.1% and 332.83 respectively). Six compounds were screened out from NCI, Enamine and Pubchem database. After molecular dynamics and binding free energy calculation, two compounds had great potential as novel CDK2 inhibitors and they also showed selective inhibition against CDK2 in the kinase activity assay.

Identification of Novel PI3Kδ Selective Inhibitors by SVM-Based Multistage Virtual Screening and Molecular Dynamics Simulations.

Phosphoinositide 3 kinase delta (PI3Kδ) is a lipid kinase that has been implicated in a variety of immune mediated disorders. The research on isoform selectivity was crucial for reducing side effects. In the current study, an optimized hierarchical multistage virtual screening method was utilized for screening the PI3Kδ selective inhibitors. The method sequentially applied a support vector machine (SVM), a protein ligand interaction fingerprint (PLIF) pharmacophore, and a molecular docking approach. The evaluation of the validation set showed a high hit rate and a high enrichment factor of 75.1% and 301.66, respectively. This multistage virtual screening method was then utilized to screen the NCI database. From the final hit list, Compound 10 has great potential as the PI3Kδ inhibitor with micromolar inhibition in the PI3Kδ kinase activity assay. This compound also shows selectivity against PI3Kδ kinase. The method combining SVM, pharmacophore, and docking was capable of screening out the compounds with potential PI3Kδ selective inhibitors. Moreover, structural modification of Compound 10 will contribute to investigating the novel scaffold and designing novel PI3Kδ inhibitors.

Exploring the mechanism of clear cell renal cell carcinoma metastasis and key genes based on multi-tool joint analysis.

Clear cell renal cell carcinoma (ccRCC) is a highly invasive urological malignant tumor that results in shorter patient survival. At present, the mechanism of ccRCC metastasis is not clear. We explored the possible mechanisms of ccRCC metastasis by analyzing the transcriptome of ccRCC patients from the Cancer Genome Atlas (TCGA) database. Comparing the differences in transcriptome in patients with and without metastasis, we found 323 differential genes (|log2FoldChange| > 1 and P < 0.001). KEGG and GO enrichment analyses of differentially expressed genes (DEGs) suggest that the transfer mechanism of ccRCC may be related to complement and coagulation cascades and cholesterol metabolism. To explore the key genes affecting tumor metastasis, we analyzed the association of these genes with patient survival time and found that 16 genes were significantly associated (P < 0.05). We compared the differences in expression of these 16 genes between ccRCC patients and the normal population, and the results showed that TF and B4GALNT1 were overexpressed in patients. Co-expression gene analysis indicated that TF may participate in the metastasis of cancer through the complement system and mucopolysaccharide biosynthesis. B4GALNT1 may affect metastasis through focal adhesion, calcium signaling pathways, and Hippo signaling pathways. Our studies suggest that the complement system and the coagulation cascade, cholesterol metabolism, calcium pathway and iron transport may be associated in the mechanism of metastasis. TF and B4GALNT1 may be the key genes for metastasis, and they may be potential diagnostic markers and therapeutic targets for ccRCC.