Lewis Antigens Research Articles

Fucosyltransferase VII promotes proliferation via the EGFR/AKT/mTOR pathway in A549 cells.

Fucosyltransferase VII (FUT7) is one of a1,3-fucosyltransferases family that catalyzes the final fucosylation step in the synthesis of Lewis antigens and generates a unique glycosylated product sialyl Lewis X (sLeX). sLeX can serve as ligands for E- or P-selectin expressed on the cell surface and results in cancer metastasis and angiogenesis. However, the molecular biological mechanisms of FUT7 elevation in neoplastic cells are still largely unknown. In this study, we examined the impact of FUT7 on cell proliferation and migration in A549 cells by colony formation assay, cell cycle assay, gelatin zymography, wound-healing assay, transwell invasion assay and Western blot. In addition, we identified that FUT7 activated EGFR/AKT/mTOR signal pathway that correlated with sLeX augmentation. In conclusion, FUT7 overexpression augments sLeX synthesis to trigger cell proliferation via the activation of EGFR/AKT/mTOR signaling pathway, which indicated that FUT7 may be a potential therapeutic target for epithelial cancers with a high expression of FUT7 and sLeX.

Microbial F-type lectin domains with affinity for blood group antigens

F-type lectins are fucose binding lectins with characteristic fucose binding and calcium binding motifs. Although they occur with a selective distribution in viruses, prokaryotes and eukaryotes, most biochemical studies have focused on vertebrate F-type lectins. Recently, using sensitive bioinformatics search techniques on the non-redundant database, we had identified many microbial F-type lectin domains with diverse domain organizations. We report here the biochemical characterization of F-type lectin domains from Cyanobium sp. PCC 7001, Myxococcus hansupus and Leucothrix mucor. We demonstrate that while all these three microbial F-type lectin domains bind to the blood group H antigen epitope on fucosylated glycans, there are fine differences in their glycan binding specificity. Cyanobium sp. PCC 7001 F-type lectin domain binds exclusively to extended H type-2 motif, Myxococcus hansupus F-type lectin domain binds to B, H type-1 and Lewisb motifs, and Leucothrix mucor F-type lectin domain binds to a wide range of fucosylated glycans, including A, B, H and Lewis antigens. We believe that these microbial lectins will be useful additions to the glycobiologist's toolbox for labeling, isolating and visualizing glycans.

Genetic Susceptibility to Rotavirus Gastroenteritis and Vaccine Effectiveness in Taiwanese Children

The genetic susceptibility to and vaccine effectiveness against rotavirus gastroenteritis were different in distinct ethnic groups. The case-control study was aimed to evaluate the effectiveness of rotavirus vaccines and associations between the histo-blood group antigens and susceptibility to rotavirus infections in a Taiwanese population. Cases were children <18 years old who were hospitalized because of laboratory-confirmed rotavirus infection. Controls were healthy children matched to cases by age and gender. The secretor status and Lewis antigen and ABO types were determined by molecular methods. A total of 68 cases and 133 controls were included. Rotavirus immunization was recorded in 8 (12%) cases and 77 (58%) controls, indicating a vaccine effectiveness of 90.3% (95% confidence interval [CI], 78.1% − 95.7%). The secretor and Lewis-positive genotypes were independently associated with increased risk of rotavirus infections (matched odds ratio [mOR] 28.5, 95% CI 2.94–277, P = 0.003 and mOR 16.8, 95% CI 1.08–2601, P = 0.04, respectively). The distribution of ABO blood types did not differ significantly between cases and controls (P = 0.47). In conclusion, Taiwanese children with the secretor genotype and Lewis-positive genotype were at increased risk of moderate-to-severe rotavirus infections. The illness can be effectively prevented by immunization in this population.

Lewis x Antigen is Associated to Head and Neck Squamous Cell Carcinoma Survival.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive disease with poor prognosis without appropriate prognostic markers. Previous research shows that Lewis antigens have been involved in carcinoma dissemination and patients´ survival. Fucosyl and sialyltransferases are the enzymes implicated in the Lewis antigens synthesis. The purpose of this study was to evaluate the prognostic utility of Lewis antigens in HNSCC. We conducted a prospective research including histological samples from 79 patients with primary HNSCC. Lewis x and sialyl Lewis x expression were detected by immunohistochemistry; patient's data, progression free, and overall survival were documented. A statistical correlation study of antigenic expression and patients´ histopathological variables was performed. Cox regression models with internal validation procedures were employed to analyze survival data. By immunohistochemistry, Lewis x was detected in 34/79 (43%) tumor samples, while sialyl Lewis x only in 11/79 (14%). Lewis x expression showed a positive correlation with tumor differentiation and a better overall survival for Lewis x+patients was detected. Moreover, multivariate Cox's regression analysis showed that Lewis x is an independent predictor of better overall survival. The in silico analysis supported the presence of deregulated fucosyl (FUT4) and sialyltransferase (ST3GAL4) in the Lewis synthetic pathway related to patient survival. These results suggest that Lewis x expression is associated with a better outcome in patients with HNSCC.

Lewis(y) antigen promotes the progression of epithelial ovarian cancer by stimulating MUC1 expression.

MUC1 is a type I transmembrane glycoprotein and is overexpressed in various epithelial tumor tissues. Some researchers have demonstrated that the glycosylation status of MUC1 can affect MUC1-mediated tumor growth and cell differentiation. In our previous study, we proved that the abilities of cell proliferation, adhesion, invasion and metastasis, and drug resistance were enhanced in ovarian cancer cells stably expressing Lewis(y). Therefore, we hypothesized that Lewis(y) antigen may play a central role in regulating MUC1 expression, and MUC1-mediated cell growth and differentiation may be closely associated with Lewis(y) antigen. This study aimed to examine the correlation between MUC1 expression and Lewis(y) antigen levels in ovarian cancer cell lines and tissue samples. A series of techniques, including RT-qPCR, western blot anlaysis, immunoprecipitation, immunohistochemistry and double-labeling immunofluorescence were applied to detect the expression of Lewis(y) and MUC1. In malignant epithelial ovarian tumors, the positive expression rates of Lewis(y) antigen and MUC1 were 88.33 and 86.67%, respectively, which were markedly higher than those in borderline (60.00 and 53.33%, P<0.05), benign (33.33 and 30%, P<0.01) and normal (0 and 25%, P<0.01) ovarian samples. There was no correlation between the positive expression rates of Lewis(y) or MUC1 and clinicopathological parameters in ovarian cancers (P>0.05). The expression levels of Lewis(y) and MUC1 correlated with the clinical FIGO stage (P<0.05). Both MUC1 and Lewis(y) were highly expressed in ovarian cancer tissues, and their expression levels were positively correlated (P<0.01). In α1,2-fucosyltransferase (α1,2-FT)-transfected cells, the gene and protein expression levels of MUC1 were significantly upregulated compared with the cells that did not overexpress α1,2-FT (P<0.05). The ratio of Lewis(y) immunoprecipitated with MUC1 to total MUC1 increased 1.55-fold in α1,2-FT-overexpressing cells (P<0.05). The overexpression of Lewis(y) resulted in the upregulation of MUC1. On the whole, our data indicate that both MUC1 and Lewis(y) are associated with the occurrence and development of ovarian cancers.

A novel O -fucosylation strategy preactivated by ( p -Tol) 2 SO/Tf 2 O and its application for the synthesis of Lewis blood group antigen Lewis a

Abstract Based on a preactivation strategy using ( p -Tol) 2 SO/Tf 2 O, a new O -fucosylation method with thioglycoside as donor under mild conditions was reported. High yields and excellent α-stereoselectivities of the fucosylation were obtained with secondary sugar alcohol as acceptors. Moreover, the novel fucosylation strategy was successfully applied to the synthesis of an important Lewis blood group antigen Lewis a , which was obtained in nine linear steps with 27% overall yield.

Helicobacter pylori Strains and Gastric MALT Lymphoma.

This article summarizes the main findings concerning Helicobacter pylori associated with gastric MALT lymphoma (GML). Considered together, GML strains based on their virulence factor profile appear to be less virulent than those associated with peptic ulcers or gastric adenocarcinoma. A particular Lewis antigen profile has been identified in GML strains and could represent an alternative adaptive mechanism to escape the host immune response thereby allowing continuous antigenic stimulation of infiltrating lymphocytes.

Genetic Susceptibility to Norovirus GII.4 Sydney Strain Infections in Taiwanese Children.

A comprehensive evaluation of associations between the susceptibility to norovirus infections and histo-blood group antigens is not available in the Taiwanese population, in which the nonsecretor phenotype is absent. A 1:1 matched case-control study was conducted in northern Taiwan from February 2013 to December 2014 when an epidemic of norovirus infection occurred. Cases were children <18 years old who were hospitalized because of diarrhea and were found to have laboratory-confirmed norovirus infections. Controls were healthy children matched to the cases by age and gender. The norovirus genotype was determined by polymerase chain reaction sequencing of the VP1 gene. The secretor status, Lewis antigen and ABO type were determined by characterization of genetic polymorphisms in the FUT2, FUT3 and ABO genes, respectively. A total of 147 case-control pairs were included. GII.4 Sydney strain was the major genotype and identified in 78.3% of the cases. The weak-secretor and Lewis-positive genotypes were less commonly identified in cases than in controls (5.4% vs. 23.1% and 79.6% vs. 89.8%, respectively). Multivariate analysis revealed that the secretor and Lewis-negative genotypes were both independent factors associated with increased risk of norovirus infections [matched odds ratio: 6.766, 95% confidence interval (CI): 2.649-17.285, P < 0.0001 and matched odds ratio: 3.071, 95% confidence interval [CI]: 1.322-7.084, P = 0.0085, respectively]. The ABO types were not significantly related to norovirus infections (P > 0.05). The weak-secretor genotype and the Lewis antigen-positive genotype were both protective factors against severe norovirus gastroenteritis during the GII.4 Sydney strain epidemic in Taiwan.

The redefinition of Helicobacter pylori lipopolysaccharide O-antigen and core-oligosaccharide domains.

Helicobacter pylori lipopolysaccharide promotes chronic gastric colonisation through O-antigen host mimicry and resistance to mucosal antimicrobial peptides mediated primarily by modifications of the lipid A. The structural organisation of the core and O-antigen domains of H. pylori lipopolysaccharide remains unclear, as the O-antigen attachment site has still to be identified experimentally. Here, structural investigations of lipopolysaccharides purified from two wild-type strains and the O-antigen ligase mutant revealed that the H. pylori core-oligosaccharide domain is a short conserved hexasaccharide (Glc-Gal-DD-Hep-LD-Hep-LD-Hep-KDO) decorated with the O-antigen domain encompassing a conserved trisaccharide (-DD-Hep-Fuc-GlcNAc-) and variable glucan, heptan and Lewis antigens. Furthermore, the putative heptosyltransferase HP1284 was found to be required for the transfer of the third heptose residue to the core-oligosaccharide. Interestingly, mutation of HP1284 did not affect the ligation of the O-antigen and resulted in the attachment of the O-antigen onto an incomplete core-oligosaccharide missing the third heptose and the adjoining Glc-Gal residues. Mutants deficient in either HP1284 or O-antigen ligase displayed a moderate increase in susceptibility to polymyxin B but were unable to colonise the mouse gastric mucosa. Finally, mapping mutagenesis and colonisation data of previous studies onto the redefined organisation of H. pylori lipopolysaccharide revealed that only the conserved motifs were essential for colonisation. In conclusion, H. pylori lipopolysaccharide is missing the canonical inner and outer core organisation. Instead it displays a short core and a longer O-antigen encompassing residues previously assigned as the outer core domain. The redefinition of H. pylori lipopolysaccharide domains warrants future studies to dissect the role of each domain in host-pathogen interactions. Also enzymes involved in the assembly of the conserved core structure, such as HP1284, could be attractive targets for the design of new therapeutic agents for managing persistent H. pylori infection causing peptic ulcers and gastric cancer.

Triazole-linked fluorescent bisboronic acid capable of selective recognition of the Lewis Y antigen

Cell surface carbohydrates of the Lewis blood group antigens, Lewis X (Lex), Lewis Y (Ley), Lewis A (Lea), and their sialylated derivatives, such as sialy Lewis X (sLex) and sialy Lewis A (sLea), play important roles in various recognition processes. These cell surface carbohydrates have also been associated with the development and progression of many types of cancers. Recently, we synthesized four anthracene-based fluorescent bisboronic acid sensors (compounds 2a–d) linked by ‘click’ chemistry with tethers of different lengths to match the epitope of various Lewis group of sugars. Among the four compounds, 2a appears to have both high sensitivity and selectivity for Ley among other carbohydrate antigens.

Human a-L-fucosidase-1 attenuates the invasive properties of thyroid cancer.

Glycans containing α-L-fucose participate in diverse interactions between cells and extracellular matrix. High glycan expression on cell surface is often associated with neoplastic progression. The lysosomal exoenzyme, α-L-fucosidase-1 (FUCA-1) removes fucose residues from glycans. The FUCA-1 gene is down-regulated in highly aggressive and metastatic human tumors. However, the role of FUCA-1 in tumor progression remains unclear. It is speculated that its inactivation perturbs glycosylation of proteins involved in cell adhesion and promotes cancer. FUCA-1 expression of various thyroid normal and cancer tissues assayed by immunohistochemical (IHC) staining was high in normal thyroids and papillary thyroid carcinomas (PTC), whereas it progressively decreased in poorly differentiated, metastatic and anaplastic thyroid carcinomas (ATC). FUCA-1 mRNA expression from tissue samples and cell lines and protein expression levels and enzyme activity in thyroid cancer cell lines paralleled those of IHC staining. Furthermore, ATC-derived 8505C cells adhesion to human E-selectin and HUVEC cells was inhibited by bovine α-L-fucosidase or Lewis antigens, thus pointing to an essential role of fucose residues in the adhesive phenotype of this cancer cell line. Finally, 8505C cells transfected with a FUCA-1 containing plasmid displayed a less invasive phenotype versus the parental 8505C. These results demonstrate that FUCA-1 is down-regulated in ATC compared to PTC and normal thyroid tissues and cell lines. As shown for other human cancers, the down-regulation of FUCA-1 correlates with increased aggressiveness of the cancer type. This is the first report indicating that the down-regulation of FUCA-1 is related to the increased aggressiveness of thyroid cancer.

Prevalence of Lewis Blood Group among Bangladeshi Population

Background: The Lewis system differs from all other blood group systems. It is primarily a system of soluble antigens originate in the tissue of body secretions, such as saliva (glycoprotein) and in plasma (glycolipid) respectively.The ABH secretor status influences red cells of Lewis phenotype and this phenotype may be modified by the ABO phenotype.Methodology: A cross sectional study was done to determine the presence of Lewis antigens in patients and voluntary donor's blood and observe the agglutination pattern during blood cross matching. In this study 350 blood samples were tested by hemagglutination -monoclonal antibodies.Result: Our study showed, Lewis antibody was detected in 50.8 % cases. The presence of anti-Leb was more frequent (69.7%) than anti-Lea in association with ABO blood group. Lewis anti-Leb was most common in O group (75.0%) followed by group-A (69.0%) where as anti-Lea was predominant in B (37.7%) followed by AB (33.3%).Moreover among Lewis phenotypes, Le (a-b+) was present in 35.4% cases, Le (a+ b-) in 15.1% and Le (a+ b+) was rare (0.3%). Phenotype Le (a-b-) represents all negative cases 172 in number (49.2%).Conclusion: Presence of Lewis antibody cause irregular agglutination interfering with interpretation of compatibility testing. So the patients with multiple transfusions or having atypical or irregular antibodies should routinely test for Lewis blood group antibodies to avoid untoward reactions.Anwer Khan Modern Medical College Journal Vol. 7, No. 2: Jul 2016, P 12-15

Identification of Lewis and Blood Group Carbohydrate Epitopes by Ion Mobility-Tandem-Mass Spectrometry Fingerprinting.

Glycans have several elements that contribute to their structural complexity, involving a range of monosaccharide building blocks, configuration of linkages between residues and various degrees of branching on a given structure. Their analysis remains challenging and resolving minor isomeric variants can be difficult, in particular terminal fucosylated Lewis and blood group antigens present on N- and O-glycans. Accurately characterizing these isomeric structures by current techniques is not straightforward and typically requires a combination of methods and/or sample derivatization. Yet the ability to monitor the occurrence of these epitopes is important as structural changes are associated with several human diseases. The use of ion mobility-mass spectrometry (IM-MS), which separates ions in the gas phase based on their size, charge and shape, offers a new potential tool for glycan analysis and recent reports have demonstrated its potential for glycomics. Here we show that Lewis and blood group isomers, which have identical fragmentation spectra, exhibit very distinctive IM drift times and collision cross sections (CCS). We show that IM-MS/MS analysis can rapidly and accurately differentiate epitopes from parotid gland N-glycans and milk oligosaccharides based on fucosylated fragment ions with characteristic CCSs.

MeCP2 regulated glycogenes contribute to proliferation and apoptosis of gastric cancer cells.

Aberrant glycogene and glycan expression is intimately associated with carcinogenesis, invasion, and metastasis of gastric cancer (GC); however the regulatory mechanisms for glycogenes in GC cells remain unclear. Methyl-CpG-binding protein 2 (MeCP2) regulates genes by binding to methylated promoters, and in our previous work we found that it is overexpressed in GC cell lines and tissues, functioning as an oncogene. In this study we detected the expression of 212 glycogenes in MeCP2 silenced GC cells versus control using the Agilent Whole Human Genome Microarray and mining the data through bioinformatic analysis. A total of 10 glycogenes exhibited increased expression (FC ≥ 2, P < 0.05), while 16 showed decreased expression (FC ≤ 2, P < 0.05) in the MeCP2 silenced cells, which corresponded to down-regulation of Lewis antigens (UEA-I), T/Tn antigens (PNA), and mature N-glycans (PHA-E and PHA-E+L) and up-regulation of lactosylceramide, a precursor oligosaccharide of N-glycans. Examination of the TCGA Gastric Cancer databases demonstrated that nine glycogenes (24.6%) were oppositely regulated by MeCP2 in MeCP2 knockdown BGC-823 cells relative to their expression level in GC tissues, and might be downstream genes of MeCP2. Individual gene analysis suggested that neutral alpha-glucosidase AB (GANAB) knockdown can rescue the effects of MeCP2 overexpression on GC cells. MeCP2 promotes GANAB by binding to the second methylated CpG island (206 bp, -12916 to -13122) of the GANAB promoter. In conclusion, glycogenes can be either up- or down-regulated by MeCP2 directly or indirectly to alter the glycopatterning and affect the proliferation and apoptosis of GC cells.

A Young Female with Difficulty in Blood Group Determination

Cold agglutinin disease (CAD) usually develops as a result of the production of a specific immunoglobulin M auto-antibody directed against the I/i and H antigens, precursors of the ABH and Lewis blood group substances, on red blood cells. Where most of the cases of cold agglutination disease usually present with features of hemolysis, acrocyanosis, respiratory symptoms or fatigue due to anemia, here we report an interesting case of CAD with unusual presentation.This 26 years female came with fever for 10 days,jaundice for two months and failure in blood group determination for blood transfusion for her anemia.The physical examination showed severe pallor, jaundice, hepatosplenomegaly and fundoscopic examination revealed Roth spots, hemorrhages and bilateral papilloedema with otherwise normal neurological examination.Complete blood count revealed severe anemia, spuriously raised MCV and MCHC with raised ESR. Peripheral blood film features were suggestive of autoimmune hemolytic anemia (Cold agglutinin disease). MRI of brain was normal. It further indicates the various manifestation of disease as in here presenting as papilloedema in patients with cold agglutination diseaseJ MEDICINE January 2017; 18 (1) : 39-41

Compositional Analysis and Metabolism of Human Milk Oligosaccharides in Infants.

It is a great success that biotechnological means are available today to produce amounts of single human milk oligosaccharides (HMOs) in a purity which allows performing metabolic and functional studies even in humans. As recent data indicate that there is a link between the Lewis blood group and the secretor status of an individual and certain inflammatory diseases, this review will also focus on the metabolic fate of secretor- and Lewis blood group-specific components. We conclude that there is no simple urinary or fecal excretion pattern of HMOs, although the pattern in urine often reflects the mother's secretor/nonsecretor status. However, there are deviations for single HMOs which deserve special attention. In feces, the variation in excretion is much higher than in urine, which may be caused by variations in the infant's intestinal microbiota. A gradual decrease in HMO excretion with time as proposed earlier does not take place as even after 7 months of exclusive breastfeeding often intact HMOs can be detected in feces and urine. In addition, we found that whenever oligosaccharides were detected in feces, LNT, the major core structure of HMOs, was present. Hence, our data do not support speculations that LNT is a preferable source for the microbiota.

H. pylori α1-3/4-fucosyltransferase (Hp3/4FT)-catalyzed one-pot multienzyme (OPME) synthesis of Lewis antigens and human milk fucosides.

Helicobacter pylori α1-3/4-fucosyltransferase (Hp3/4FT) was expressed in Escherichia coli at a level of 30 mg L-1 culture and used as a diverse catalyst in a one-pot multienzyme (OPME) system for high-yield production of l-fucose-containing carbohydrates including Lewis antigens such as Lewis a, b, and x, O-sulfated Lewis x, and sialyl Lewis x and human milk fucosides such as 3-fucosyllactose (3-FL), lacto-N-fucopentaose (LNFP) III, and lacto-N-difuco-hexaose (LNDFH) II and III. Noticeably, while difucosylation of tetrasaccharides was readily achieved using an excess amount of donor, the synthesis of LNFP III was achieved by Hp3/4FT-catalyzed selective fucosylation of the N-acetyllactosamine (LacNAc) component in lacto-N-neotetraose (LNnT).

Automated Glycan Assembly of Oligo-N-Acetyllactosamine and Keratan Sulfate Probes to Study Virus-Glycan Interactions

Summary Oligo- N -acetyllactosamine (LacNAc) and keratan sulfate (KS) glycans exert crucial functions in disease-relevant processes, including cancer formation, inflammation, and viral infection. To facilitate structure-activity studies with these glycans, we established a universal strategy to synthesize linear and branched LacNAc as well as differentially sulfated KS oligosaccharides by automated glycan assembly. We synthesized oligosaccharides as long as hexamers by combining four monosaccharide building blocks. Key to the strategy was installing three orthogonal protection groups, 9-fluorenylmethoxycarbonyl (Fmoc), levulinoyl (Lev) ester, and 2-naphthylmethyl (Nap) ether, which were selectively removed from a common oligosaccharide precursor for differential sulfation. Microarrays presenting the synthetic oligosaccharides revealed a specific interaction between a disulfated KS tetrasaccharide and the adeno-associated virus AAVrh10 gene-therapy vector, which was further corroborated by surface plasmon resonance studies. Thus, KS represents a novel receptor candidate for AAVrh10. These insights could have implications for cell-type-specific gene-delivery approaches.

Crude Preparation of Lipopolysaccharide from Helicobacter pylori for Silver Staining and Western Blot.

This protocol provides an easy and rapid method to prepare lipopolysaccharide from the gastric pathogen Helicobacter pylori for visualization on acrylamide gels by silver staining and for detecting the presence of Lewis antigens by Western blot. The silver staining is a four-step procedure, involving a 20 min-oxidation step, a 10 min-silver staining step, a 2-10 min color development step and finally a 1-min color termination step. Lipopolysaccharide from H. pylori wild-type and corresponding mutants analyzed by this method are described in a recent publication (Li et al., 2017). This crude preparation of LPS for silver staining is also applicable in other Gram-negative bacteria.

FUT2 genetic variants as predictors of tumor development with hepatocellular carcinoma.

Lewis antigens related to the ABO blood group are fucosylated oligosaccharides and are synthesized by specific glycosyltransferases (FUTs). FUTs are involved in various biological processes including cell adhesion and tumor progression. The fucosyltransferase-2 gene (FUT2) encodes alpha (1,2) fucosyltransferase, which is responsible for the addition of the alpha (1,2)-linkage of fucose to glycans. Aberrant fucosylation occurs frequently during the development and progression of hepatocellular carcinoma (HCC). However, the association of FUT2 polymorphisms with HCC development has not been studied. Therefore, we aimed to investigate the association of FUT2 polymorphisms with demographic, etiological, and clinical characteristics and with susceptibility to HCC. In this study, a total of 339 patients and 720 controls were recruited. The genotypes of FUT2 at four single-nucleotide polymorphisms (SNPs; rs281377, rs1047781, rs601338, and rs602662) were detected by real-time polymerase chain reaction from these samples. Compared with the wild-type genotype at SNP rs1047781, which is homozygous for nucleotides AA, at least one polymorphic T allele (AT or TT) displayed significant association with clinical stage (p = 0.048) and tumor size (p = 0.022). Our study strongly implicates the polymorphic locus rs1047781 of FUT2 as being associated with HCC development.