hTERT promotes the invasion of gastric cancer cells by enhancing FOXO3a ubiquitination and subsequent ITGB1 upregulation.

BACKGROUND AND AIMS: Human telomerase reverse transcriptase (hTERT) plays an important role in cancer invasion, but the relevant mechanism is not well known. This study aims to investigate the role and mechanism of hTERT in gastric cancer metastasis. DESIGN: Proteomics analysis, qPCR and western blotting were used to screen for hTERT-regulated candidate molecules in gastric cancer invasion. Chromatin immunoprecipitation (ChIP) qPCR was performed to identify the binding sites of hTERT at the regulatory region of the integrin ß1 (ITGB1) gene. ChIP assays were further applied to elucidate the transcription factors that bound to the regulatory region. The interactions between hTERT and the transcription factors were tested by co-immunoprecipitation (Co-IP) and glutathione S-transferase (GST) pull-down experiments. Moreover, the revealed pathway was verified in tumour-bearing nude mice and human gastric cancer tissues. RESULTS: ITGB1 was identified as a downstream gene of hTERT, and there were two hTERT-binding regions within this gene. hTERT alleviated the binding of forkhead box O3 (FOXO3a) to FOXO3a binding element (+9972∼+9978), but it enhanced the binding of forkhead box M1 (FOXM1) to FOXM1 binding element (-1104∼-1109) in ITGB1 gene. Importantly, FOXO3a played a major role in hTERT-induced ITGB1 expression, and the hTERT/murine double minute 2 (MDM2) complex promoted the ubiquitin-mediated degradation of FOXO3a. Moreover, hTERT increased ITGB1 expression in xenograft gastric cancer, and the level of hTERT was positively correlated with that of ITGB1 in human gastric cancer tissues. CONCLUSIONS: The hTERT/MDM2-FOXO3a-ITGB1 pathway markedly contributes to hTERT-promoted gastric cancer invasion, suggesting that this pathway might be a novel target for the prevention and treatment of gastric cancer metastasis.

Population Relationship of Vibrio parahaemolyticus Isolates Derived from Aquaculture Ponds, a Seafood Market, Restaurants, and Clinical Samples.

To study the relationship between environmental and clinical populations of Vibrio parahaemolyticus, we collected in total 86 isolates from Southern China during one and a half years. Sixty-eight isolates were recovered from aquaculture ponds, a seafood market, and restaurants, and 18 isolates were recovered from clinical samples. Virulence gene analysis revealed that 25 isolates (14 clinical and 11 environmental) tested positive for tdh, but only 4 carried trh. Interestingly, none of the tdh(+) environmental isolates was recovered from ponds. Both environmental and clinical tdh(+) isolates, except for one clinical isolate, harbor type III secretion system 2α (T3SS2α) and T3SS2ß-related genes, including vopB2α, which was previously suggested to be absent from environmental strains. More than 70% of clinical isolates carried the pandemic marker of new toxRS (GS-PCR(+)), which was not present in the environmental isolates. Pulsed-field gel electrophoresis and multilocus sequence typing analysis showed a high degree of genetic diversity within the environmental isolates. In contrast, the clinical population formed a tight cluster that differed from the environmental isolates. These findings suggest that the pandemic strains of V. parahaemolyticus may not directly originate from marine animals. Rather the environments where they are maintained could serve as reservoirs for toxigenic, but not pandemic strains. These environments provide an ideal place for generation of new toxigenic strains through DNA exchange, which was revealed by extensive recombination events in recA sequences of the environmental isolates.

A pro-inflammatory role for Th22 cells in Helicobacter pylori-associated gastritis.

OBJECTIVE: Helper T (Th) cell responses are critical for the pathogenesis of Helicobacter pylori-induced gastritis. Th22 cells represent a newly discovered Th cell subset, but their relevance to H. pylori-induced gastritis is unknown. DESIGN: Flow cytometry, real-time PCR and ELISA analyses were performed to examine cell, protein and transcript levels in gastric samples from patients and mice infected with H. pylori. Gastric tissues from interleukin (IL)-22-deficient and wild-type (control) mice were also examined. Tissue inflammation was determined for pro-inflammatory cell infiltration and pro-inflammatory protein production. Gastric epithelial cells and myeloid-derived suppressor cells (MDSC) were isolated, stimulated and/or cultured for Th22 cell function assays. RESULTS: Th22 cells accumulated in gastric mucosa of both patients and mice infected with H. pylori. Th22 cell polarisation was promoted via the production of IL-23 by dendritic cells (DC) during H. pylori infection, and resulted in increased inflammation within the gastric mucosa. This inflammation was characterised by the CXCR2-dependent influx of MDSCs, whose migration was induced via the IL-22-dependent production of CXCL2 by gastric epithelial cells. Under the influence of IL-22, MDSCs, in turn, produced pro-inflammatory proteins, such as S100A8 and S100A9, and suppressed Th1 cell responses, thereby contributing to the development of H. pylori-associated gastritis. CONCLUSIONS: This study, therefore, identifies a novel regulatory network involving H. pylori, DCs, Th22 cells, gastric epithelial cells and MDSCs, which collectively exert a pro-inflammatory effect within the gastric microenvironment. Efforts to inhibit this Th22-dependent pathway may therefore prove a valuable strategy in the therapy of H. pylori-associated gastritis.

Long Noncoding RNA in Digestive Tract Cancers: Function, Mechanism, and Potential Biomarker.

Digestive tract cancers (DTCs) are a leading cause of cancer-related death worldwide. Current therapeutic tools for advanced stage DTCs have limitations, and patients with early stage DTCs frequently have a missed diagnosis due to shortage of efficient biomarkers. Consequently, it is necessary to develop novel biomarkers for early diagnosis and novel therapeutic targets for treatment of DTCs. In recent years, long noncoding RNAs (lncRNAs), a class of noncoding RNAs with >200 nucleotides, have been shown to be aberrantly expressed in DTCs and to have an important role in DTC development: the expression profiles of lncRNAs strongly correlated with poor survival of patients with DTCs, and lncRNAs acted as oncogenes or tumor suppressor genes in DTC progression. In this review, we summarized the functional lncRNAs and expounded on their regulatory mechanisms in DTCs.

Helicobacter pylori virulence factor CagA promotes tumorigenesis of gastric cancer via multiple signaling pathways.

Helicobacter pylori (H. pylori) infection is strongly associated with the development of gastric diseases but also with several extragastric diseases. The clinical outcomes caused by H. pylori infection are considered to be associated with a complex combination of host susceptibility, environmental factors and bacterial isolates. Infections involving H. pylori strains that possess the virulence factor CagA have a worse clinical outcome than those involving CagA-negative strains. It is remarkable that CagA-positive H. pylori increase the risk for gastric cancer over the risk associated with H. pylori infection alone. CagA behaves as a bacterial oncoprotein playing a key role in H. pylori-induced gastric cancer. Activation of oncogenic signaling pathways and inactivation of tumor suppressor pathways are two crucial events in the development of gastric cancer. CagA shows the ability to affect the expression or function of vital protein in oncogenic or tumor suppressor signaling pathways via several molecular mechanisms, such as direct binding or interaction, phosphorylation of vital signaling proteins and methylation of tumor suppressor genes. As a result, CagA continuously dysregulates of these signaling pathways and promotes tumorigenesis. Recent research has enriched our understanding of how CagA effects on these signaling pathways. This review summarizes the results of the most relevant studies, discusses the complex molecular mechanism involved and attempts to delineate the entire signaling pathway.

A dominant CD4(+) T-cell response to Helicobacter pylori reduces risk for gastric disease in humans.

BACKGROUND & AIMS: Immunodominance is an important feature of antiviral, antitumor, and antibacterial cellular immune responses, but it is not well demonstrated in the immune responses against Helicobacter pylori. Antigen-specific CD4(+) T cells protect mice against infection with H pylori. We investigated the immunodominant CD4(+) T-cell response to neuraminyllactose-binding hemagglutinin (HpaA), which is a conserved, H pylori-specific colonization factor that is being investigated as an antigen for vaccination strategies. METHODS: HpaA-specific CD4(+) T cells were expanded with autologous peripheral blood mononuclear cells that had been incubated with recombinant HpaA and characterized using overlapping synthetic peptides. We compared the percentage of CD4(+) T cells with specificity for HpaA(88-100), restricted to HLA-DRB1*1501, among 59 H pylori-infected subjects with different gastric diseases. RESULTS: We identified and characterized several immunodominant CD4(+) T-cell epitopes derived from HpaA. The immunodominant CD4(+) T-cell responses specific to HpaA(88-100) were observed in most H pylori-infected individuals who expressed HLA-DRB1*1501 and were significantly more abundant in patients with less severe diseases (P < .05). CONCLUSIONS: The HLA-DRB1*1501-restricted immunodominant CD4(+) T-cell response to HpaA(88-100) is associated with reduced risk of severe gastric diseases. Further study of these and other immunodominant CD4(+) T-cell responses to H pylori will provide insight into mechanisms of protective immunity and aid in vaccine design.

The hTERT-p50 homodimer inhibits PLEKHA7 expression to promote gastric cancer invasion and metastasis.

Although accumulating evidence has highlighted the molecular mechanisms by which hTERT promotes tumour cell invasion and metastasis, the molecular mechanisms of the properties enabling hTERT to contribute to invasion and metastasis have not been clearly illustrated. Here, we report that hTERT promotes gastric cancer invasion and metastasis by recruiting p50 to synergistically inhibit PLEKHA7 expression. We observed that the expression of PLEKHA7 in gastric cancer was significantly negatively associated with the TNM stage and lymphatic metastasis and that decreased PLEKHA7 expression dramatically increased invasion and metastasis in gastric cancer cells. Further mechanistic research showed that hTERT directly regulates PLEKHA7 expression by binding p50 and recruiting the hTERT/p50 complex to the PLEKHA7 promoter. Increased hTERT dramatically decreased PLEKHA7 expression and promoted invasion and metastasis in gastric cancer cells. The hTERT-mediated invasion/metastasis properties at least partially depended on PLEKHA7. Our work uncovers a novel molecular mechanism underlying invasion/metastasis in gastric cancer orchestrated by hTERT and p50.

H. pylori related proinflammatory cytokines contribute to the induction of miR-146a in human gastric epithelial cells.

MicroRNAs have been implicated as a central regulator of the immune system. We have previously reported that Helicobacter pylori (H. pylori) was able to increase the expression of miR-146a, and miR-146a may negatively regulate H. pylori-induced inflammation, but the exact mechanism of how H. pylori contribute the induction of miR-146a is not clear. Here, we attempted to assess the role of H. pylori related proinflammatory cytokines including interleukin (IL)-8, tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß, and cytotoxin-associated gene A (CagA) virulence factor on the induction of miR-146a. We found that IL-8, TNF-α, and IL-1ß could contribute to the induction of miR-146a in gastric epithelial cell HGC-27 in NF-κB-dependent manner, while the induction of miR-146a upon H. pylori stimulation was independent of above proinflammatory cytokines. Furthermore, overexpression of miR-146a reduced H. pylori-induced IL-8, TNF-α, and IL-1ß. However, CagA had no effect on the miR-146a induction. Taken together, our study suggest that proinflammatory cytokines IL-8, TNF-α, and IL-1ß could contribute to the induction of miR-146a during H. pylori infection, while CagA is not necessarily required for miR-146a induction. miR-146a may function as novel negative regulators to modulate the inflammation.

[Surveillance and analysis on the pathogenic features of Salmonella in Guangdong province in 2010].

OBJECTIVE: In order to better understand the nature of Salmonella infection in diarrheal patients in Guangdong province, the study analyzed the serum types, antibiotic resistance and molecular determinants of the isolated Salmonella strains. METHODS: In year 2010, 8405 diarrhea patients from 16 surveillant hospital in Guangzhou, Zhongshan, Dongguan, Zhuhai, Maoming, Yangjiang and Jiangmen cities in Guangdong province, were recruited in the study. A total of 8405 fecal specimen were collected and subjected to Salmonella isolation and culture. The isolated Salmonella strains were further analyzed via serotyping, antimicrobial susceptibility testing, and PFGE. The χ(2) test was applied to compare the differences between the isolated Salmonella strains in different seasons and districts. BioNumerics software was used to analyze the PFGE results in order to determine the correlation between different Salmonella strains. RESULTS: The positive rate of the surveillant Salmonella in Guangdong province was 3.58% (301/8405) in 2010; with the gender ratio at 1.34:1 (166/124). Salmonella infection was found in all age groups, and most in infants, accounting for 57.48% (173/301). The isolated rates of Salmonella were separately 3.48% (61/1751), 4.97% (134/2695), 3.08% (73/2370) and 2.08% (33/1589) in the four seasons; and the difference was statistically significant (χ(2) = 27.29, P < 0.01). The isolated rates of Salmonella in different regions were as follows: Zhuhai 15.43% (25/162), Maoming 7.53% (18/239), Dongguan 6.51% (39/599), Yangjiang 3.64% (14/385), Zhongshan 3.03% (70/2309), Guangzhou 2.90% (126/4349) and Jiangmen 2.49% (9/362). The difference between regions was statistically significant (χ(2) = 100.75, P < 0.01). Except one strain of the isolated Salmonella cannot be serotyped, the other 300 strains were divided into 42 serotypes, of which Salmonella typhimurium and Salmonella enteritidis were dominant, account for 45.18% (136/301) and 10.96% (33/301) respectively. Although over 85% of Salmonella were sensitive to cephalosporin, ACSSuT resistance patterns (defined as resistance to at least ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline) reached 34.88% (105/301), the highest resistant rate was found in serotype Salmonella typhimurium, as high as 65.44% (89/136). 136 strains of Salmonella typhimurium were divided into 51 PFGE types, showed great genetic diversity. 33 strains of Salmonella enteritidis were divided into 18 PFGE types. The strains with same PFGE pattern may have different drug-resistant patterns, and vice versa. CONCLUSION: Salmonella typhimurium and Salmonella enteritidis were the dominant serotypes causing infectious diarrhea in Guangdong province. Cephalosporin was the primary choice in clinical medicine. However, Salmonella typhimurium was resistant to drug most seriously in Guangdong province. There was no significant correlation between Salmonella resistance patterns and PFGE type.

Role of lncSLCO1C1 in gastric cancer progression and resistance to oxaliplatin therapy.

BACKGROUND: Gastric carcinoma (GC) is one of the most deadly diseases due to tumour metastasis and resistance to therapy. Understanding the molecular mechanism of tumour progression and drug resistance will improve therapeutic efficacy and develop novel intervention strategies. METHODS: Differentially expressed long non-coding RNAs (lncRNAs) in clinical specimens were identified by LncRNA microarrays and validated in different clinical cohorts by quantitative real-time polymerase chain reaction (qRT-PCR), in situ hybridisation and bioinformatics analysis. Biological functions of lncRNA were investigated by using cell proliferation assays, migration assays, xenograft tumour models and bioinformatics analysis. Effects of lncSLCO1C1 on GC cell survival were assessed by comet assays and immunofluorescence assays. Underlying molecular mechanisms were further explored by using a number of technologies including RNA pull-down, mass spectrometry analysis, RNA immunoprecipitation, co-immunoprecipitation, miRNA sequencing, luciferase reporter assays and molecular modelling. RESULTS: LncSLCO1C1 was highly upregulated in GC tissue samples and associated with GC patients' poor overall survival. Overexpression of lncSLCO1C1 promoted proliferation and migration, whereas decreased lncSLCO1C1 expression produced the opposite effects. lncSLCO1C1 also mediated tumour resistance to chemotherapy with oxaliplatin by reducing DNA damage and increasing cell proliferation. Despite sequence overlapping between lncSLCO1C1 and PDE3A, alternations of PDE3A expression had no effect on the GC cell progression, indicating that lncSLCO1C1, not PDE3A, related with the progression of GC cells. Mechanistically, lncSLCO1C1 serves as a scaffold for the structure-specific recognition protein 1 (SSRP1)/H2A/H2B complex and regulates the function of SSRP1 in reducing DNA damage. Meanwhile, lncSLCO1C1 functions as a sponge to adsorb miR-204-5p and miR-211-5p that target SSRP1 mRNA, and thus increases SSRP1 expression. Patients with high expressions of both lncSLCO1C1 and SSRP1 have poor overall survival, highlighting the role of lncSLCO1C1 in GC progression. CONCLUSIONS: LncSLCO1C1 promotes GC progression by enhancing cell growth and preventing DNA damage via interacting and scaffolding the SSRP1/H2A/H2b complex and absorbing both miR-211-5p and miR-204-5p to increase SSRP1 expression.

Screening and identification of natural antisense transcripts in Helicobacter pylori by a novel approach based on RNase I protection assay.

Natural antisense transcripts (NATs) are endogenous RNA molecules that exhibit partial or complete complementarity to other RNAs. Studies have shown that NATs may participate in a broad range of gene regulatory events. The identification of NATs in human, mouse and Escherichia coli has revealed their widespread occurrence in both eukaryotic and prokaryotic life. However, little is known about NATs in Helicobacter pylori (H. pylori), a human pathogen which is associated with gastric diseases. Here we systematically screened NATs in H. pylori by a novel experimental strategy based on RNase I protection assay. We successfully constructed a cDNA library of NATs and developed a novel poly(A)-tailed RT-PCR method to monitor the expression of NATs. After sequencing, bioinformatic analysis and expression detection, two novel NATs (NAT-39 and NAT-67) were confirmed. They were, respectively, complementary to the following genes: iron-regulated outer membrane protein (frpB) and periplasmic iron-binding protein (ceuE). Taken together, the results suggest that NAT-39 and NAT-67 may participate in the regulation of iron homeostasis in H. Pylori in a sequence complementary manner with target mRNAs.

Long noncoding RNA LINC00675 enhances phosphorylation of vimentin on Ser83 to suppress gastric cancer progression.

Long noncoding RNAs (lncRNAs) play a crucial role in cancer development, but few lncRNAs have been functionally characterized in gastric cancer (GC). Here, we reported an lncRNA LINC00675 whose expression was significantly decreased in GC tissues compared with the adjacent non-tumor tissues, and its low expression was associated with the poor survival of GC patients. Gain-and loss-of-function studies indicated that LINC00675 was a tumor suppressor because it repressed the proliferation, migration and invasion of GC cells in vitro and also inhibited the distal pulmonary and hepatic metastases of GC cells in vivo. Mechanistic investigations revealed that LINC00675 interacted with vimentin, a protein involved in cell metastasis, and enhanced its phosphorylation level on Ser83 to result in the collapse of vimentin filament in GC cells, thereby reducing cell metastasis. Taken together, our findings indicate that LINC00675 expression signature may serve as a novel biomarker for the diagnosis and prognosis of GC, and also highlight that LINC00675/vimentin complex may be a potentially therapeutic target of GC.

miR-93-5p/IFNAR1 axis promotes gastric cancer metastasis through activating the STAT3 signaling pathway.

Aberrant expression of microRNAs (miRNAs) plays an important role in gastric cancer (GC) development. miR-93-5p has shown opposing functions in different types of cancers, but the exact expression pattern and molecular mechanism of miR-93-5p in GC development remain to be elucidated. Here, we reported that miR-93-5p expression was increased in GC tissues compared with the adjacent normal tissues and that its overexpression was correlated with distant metastasis and poor survival in GC patients. miR-93-5p knockdown inhibited the migration, invasion and proliferation of GC cells in vitro and in vivo, while its overexpression displayed an opposite result. Using an mRNA microarray, we found that miR-93-5p significantly downregulated IFNAR1 expression in GC cells, which was further identified as a direct target of miR-93-5p. IFNAR1 knockdown promoted GC cell migration and invasion, but its restoration could rescue GC cell migration and invasion induced by miR-93-5p overexpression. Moreover, miR-93-5p-IFNAR1 axis increased MMP9 expression via STAT3 pathway in GC cells. Taken together, we reveal that miR-93-5p overexpression is associated with the poor survival of GC patients and miR-93-5p-IFNAR1 axis promotes GC metastasis through activation of STAT3 pathway.

Ultra-sensitive colorimetric method to quantitate hundreds of polynucleotide molecules by gold nanoparticles with silver enhancement.

An ultra-sensitive colorimetric method to quantitate hundreds of polynucleotide molecules by gold nanoparticles with silver enhancement has been developed. The hybridization products from the target polynucleotides with biotin-labeled probes and gold nanoparticle-functioned oligonucleotides were immobilized to microplates via avidin-biotin system, and the absorbance signals of gold nanoparticles were amplified by silver enhance solution. This sandwich colorimetric assay can detect as few as 600 molecules for single-strand oligonucleotides and as few as 6000 molecules for double-strand polynucleotides in a 50 microL reaction system.

Long non-coding RNAs in colorectal cancer.

Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. Despite substantial progress in understanding the molecular mechanisms and treatment of CRC in recent years, the overall survival rate of CRC patients has not improved dramatically. The development of CRC is multifactor and multistep processes, in which abnormal gene expression may play an important role. With the advance of human tumor molecular biology, a series of studies have highlighted the role of long non-coding RNAs (lncRNAs) in the development of CRC. CRC-related lncRNAs have been demonstrated to regulate the genes by various mechanisms, including epigenetic modifications, lncRNA-miRNA and lncRNA-protein interactions, and by their actions as miRNA precursors or pseudogenes. Since some lncRNAs can be detected in human body fluid and have good specificity and accessibility, they have been suggested to be used as novel potential biomarkers for CRC diagnosis and prognosis as well as in the prediction of the response to therapy. Therefore, in this review, we will focus on lncRNAs in CRC development, the mechanisms and biomarkers of lncRNAs in CRC.

Peptide-Based Treatment: A Promising Cancer Therapy.

Many new therapies are currently being used to treat cancer. Among these new methods, chemotherapy based on peptides has been of great interest due to the unique advantages of peptides, such as a low molecular weight, the ability to specifically target tumor cells, and low toxicity in normal tissues. In treating cancer, peptide-based chemotherapy can be mainly divided into three types, peptide-alone therapy, peptide vaccines, and peptide-conjugated nanomaterials. Peptide-alone therapy may specifically enhance the immune system's response to kill tumor cells. Peptide-based vaccines have been used in advanced cancers to improve patients' overall survival. Additionally, the combination of peptides with nanomaterials expands the therapeutic ability of peptides to treat cancer by enhancing drug delivery and sensitivity. In this review, we mainly focus on the new advances in the application of peptides in treating cancer in recent years, including diagnosis, treatment, and prognosis.

The emergence of long non-coding RNAs in the tumorigenesis of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third cause of cancer-related death worldwide. However, the treatments for HCC are limited, and most of them are only available to the early stage. In the later stages, traditional chemotherapy has only marginal effects and may include toxicity. Thus, the identification of new predictive markers is urgently needed. New targets for non-conventional treatments will help to accelerate research on the molecular pathogenesis of HCC. A new class of transcripts, long non-coding RNAs (lncRNAs), has recently been found to be pervasively transcribed in the human genome. Aberrant expression of several lncRNAs was found to be involved in the tumorigenesis of HCC. In this review, we describe the possible molecular mechanisms that underlie lncRNA expression changes in HCC, as well as potential future applications of lncRNA research in the diagnosis and treatment of HCC.

miR-149 represses metastasis of hepatocellular carcinoma by targeting actin-regulatory proteins PPM1F.

microRNAs have been implicated in hepatocellular carcinoma (HCC) metastasis, which is predominant cause of high mortality in these patients. Although an increasing body of evidence indicates that miR-149 plays an important role in the growth and metastasis of multiple types of cancers, its role in the progression of HCC remains unknown. Here, we demonstrated that miR-149 was significantly down-regulated in HCC, which was correlated with distant metastasis and TNM stage with statistical significance. A survival analysis showed that decreased miR-149 expression was correlated with a poor prognosis of HCC as well. We found that over-expression of miR-149 suppressed migration and invasion of HCC cells in vitro. In addition, we identified PPM1F (protein phosphatase, Mg(2+)/Mn(2+)-dependent, 1F) as a direct target of miR-149 whose expression was negatively correlated with the expression of miR-149 in HCC tissues. The re-expression of PPM1F rescued the miR-149-mediated inhibition of cell migration and invasion. miR-149 regulated formation of stress fibers to inhibit migration, and re-expression of PPM1F reverted the miR-149-mediated loss of stress fibers. Moreover, we demonstrated that over-expression of miR-149 reduced pMLC2, a downstream effector of PPM1F, in MHCC-97H cells. In vivo studies confirm inhibition of HCC metastasis by miR-149. Taken together, our findings indicates that miR-149 is a potential prognostic biomarker of HCC and that the miR-149/PPM1F regulatory axis represents a novel therapeutic target for HCC treatment.