Genome-wide enhancer RNA profiling adds molecular links between genetic variation and human cancers.

BACKGROUND: Dysregulation of enhancer transcription occurs in multiple cancers. Enhancer RNAs (eRNAs) are transcribed products from enhancers that play critical roles in transcriptional control. Characterizing the genetic basis of eRNA expression may elucidate the molecular mechanisms underlying cancers. METHODS: Initially, a comprehensive analysis of eRNA quantitative trait loci (eRNAQTLs) was performed in The Cancer Genome Atlas (TCGA), and functional features were characterized using multi-omics data. To establish the first eRNAQTL profiles for colorectal cancer (CRC) in China, epigenomic data were used to define active enhancers, which were subsequently integrated with transcription and genotyping data from 154 paired CRC samples. Finally, large-scale case-control studies (34,585 cases and 69,544 controls) were conducted along with multipronged experiments to investigate the potential mechanisms by which candidate eRNAQTLs affect CRC risk. RESULTS: A total of 300,112 eRNAQTLs were identified across 30 different cancer types, which exert their influence on eRNA transcription by modulating chromatin status, binding affinity to transcription factors and RNA-binding proteins. These eRNAQTLs were found to be significantly enriched in cancer risk loci, explaining a substantial proportion of cancer heritability. Additionally, tumor-specific eRNAQTLs exhibited high responsiveness to the development of cancer. Moreover, the target genes of these eRNAs were associated with dysregulated signaling pathways and immune cell infiltration in cancer, highlighting their potential as therapeutic targets. Furthermore, multiple ethnic population studies have confirmed that an eRNAQTL rs3094296-T variant decreases the risk of CRC in populations from China (OR = 0.91, 95%CI 0.88-0.95, P = 2.92 × 10-7) and Europe (OR = 0.92, 95%CI 0.88-0.95, P = 4.61 × 10-6). Mechanistically, rs3094296 had an allele-specific effect on the transcription of the eRNA ENSR00000155786, which functioned as a transcriptional activator promoting the expression of its target gene SENP7. These two genes synergistically suppressed tumor cell proliferation. Our curated list of variants, genes, and drugs has been made available in CancereRNAQTL ( http://canernaqtl.whu.edu.cn/#/ ) to serve as an informative resource for advancing this field. CONCLUSION: Our findings underscore the significance of eRNAQTLs in transcriptional regulation and disease heritability, pinpointing the potential of eRNA-based therapeutic strategies in cancers.

CDC Like Kinase 2 plays an oncogenic role in colorectal cancer via modulating the Wnt/ß-catenin signaling.

Colorectal cancer (CRC) is a common malignant tumor with high morbidity and mortality, and significant heterogeneity among patients. In this study, we aimed to explore the role and mechanism of CLK2 in CRC, a kinase that phosphorylates SR proteins involved in splicing. Based on the analysis from The Cancer Genome Atlas (TCGA) dataset and tissue microarray, we found that CLK2 was upregulated in CRC tissues and associated with a higher tumor stage and poorer overall survival. Consistent with the bioinformatics analysis, the functional experiments validated that CLK2 acted as a tumor-promoting factor in CRC progression. CLK2 knockdown suppressed aggressive cell proliferation, migration, and invasion in vitro, as well as restrained tumor growth in vivo. In terms of mechanism, we found that the Wnt/ß-catenin signaling pathway was responsible for the CLK2-induced CRC progression, based on the results of pathway enrichment analysis and subsequent experimental validation. Thus, our study, for the first time, identified the role of CLK2 in CRC development and provided a compelling biomarker for targeted therapy in CRC treatment.

The combination of body composition conditions and systemic inflammatory markers has prognostic value for patients with gastric cancer treated with adjuvant chemoradiotherapy.

OBJECTIVE: The aim of this study was to explore the prognostic value of the association between systemic inflammation response markers (red blood cell distribution width, neutrophil platelet score, prognostic nutritional index, neutrophil-to-lymphocyte ratio, neutrophil-to-platelet ratio, lymphocyte-to-monocyte ratio, and systemic immune-inflammation index) and poorer body composition conditions (sarcopenia, myosteatosis, and sarcopenic obesity) among patients with gastric cancer who underwent adjuvant chemoradiotherapy after radical gastrectomy. METHODS: A computed tomography scan was performed within 2 wk of prechemoradiotherapy to identify sarcopenia, myosteatosis and sarcopenic obesity. Tumor and systemic inflammatory response information was recorded. Logistic analysis was used to explore the potential risk factors associated with body composition. Univariate and multivariate Cox analyses were performed for survival analysis. A nomogram was constructed to serve as a prognostic prediction tool for the 3- and 5-y overall survival rates. RESULTS: The study included 223 patients (74 women and 149 men) with gastric cancer treated with adjuvant chemoradiotherapy after radical gastrectomy. The incidences of sarcopenia, myosteatosis, and sarcopenic obesity were 30%, 39%, and 16%, respectively. Logistic analysis demonstrated that a low prognostic nutritional index is a risk factor for sarcopenia, myosteatosis, and sarcopenic obesity. Based on survival analysis, stage (hazard ratio [HR], 0.44; 95% confidence interval [CI], 0.23-0.84; P = 0.01), the neutrophil platelet score (HR, 0.50; 95% CI, 0.31-0.82; P = 0.01), the prognostic nutritional index (HR, 0.40; 95% CI, 0.24-0.68; P = 0.00) and sarcopenic obesity (HR, 0.54; 95% CI, 0.31-0.93; P = 0.03) remained independent prognostic factors for overall survival. Accuracy was improved when systemic inflammation markers were incorporated into the nomogram compared with when they were excluded, and the predicted C indexes of the nomogram with and without systemic inflammatory markers were 0.71 (95% CI, 0.67-0.73) and 0.63 (95% CI, 0.57-0.68), respectively. CONCLUSION: The systemic inflammatory response associated with progressive nutritional conditions and body composition conditions with systemic inflammation markers incorporated presented better prognostic value.

Characteristics of immune and inflammatory responses among different age groups of pediatric patients with COVID-19 in China.

BACKGROUND: Severe cases of coronavirus disease 2019 (COVID-19) among pediatric patients are more common in children less than 1 year of age. Our aim is to address the underlying role of immunity and inflammation conditions among different age groups of pediatric patients. METHODS: We recruited pediatric patients confirmed of moderate COVID-19 symptoms, admitted to Wuhan Children's Hospital from January 28th to April 1st in 2020. Patients were divided into four age groups (≤ 1, 1-6, 7-10, and 11-15 years). Demographic information, clinical characteristics, laboratory results of lymphocyte subsets test, immune and inflammation related markers were all evaluated. RESULTS: Analysis included 217/241 (90.0%) of patients with moderate clinical stage disease. Average recovery time of children more than 6 years old was significantly shorter than of children younger than 6 years (P = 0.001). Reduced neutrophils and increased lymphocytes were significantly most observed among patients under 1 year old (P < 0.01). CD19+ B cells were the only significantly elevated immune cells, especially among patients under 1 year old (cell proportion: n = 12, 30.0%, P < 0.001; cell count: n = 13, 32.5%, P < 0.001). While, low levels of immune related makers, such as immunoglobulin (Ig) G (P < 0.001), IgA (P < 0.001), IgM (P < 0.001) and serum complement C3c (P < 0.001), were also mostly found among patients under 1 year old, together with elevated levels of inflammation related markers, such as tumor necrosis factor γ (P = 0.007), interleukin (IL)-10 (P = 0.011), IL-6 (P = 0.008), lactate dehydrogenase (P < 0.001), and procalcitonin (P = 0.007). CONCLUSION: The higher rate of severe cases and long course of COVID-19 among children under 1 year old may be due to the lower production of antibodies and serum complements of in this age group.

Polycomb complex protein BMI-1 promotes invasion and metastasis of pancreatic cancer stem cells by activating PI3K/AKT signaling, an ex vivo, in vitro, and in vivo study.

Cancer stem cell theory indicates cancer stem cells are the key to promote tumor invasion and metastasis. Studies showed that BMI-1 could promote self-renew, differentiation and tumor formation of CSCs and invasion/metastasis of human cancer. However, whether BMI-1 could regulate invasion and metastasis ability of CSCs is still unclear. In our study, we found that up-regulated expression of BMI-1 was associated with tumor invasion, metastasis and poor survival of pancreatic cancer patients. CD133+ cells were obtained by using magnetic cell sorting and identified of CSCs properties such as self-renew, multi-differentiation and tumor formation ability. Then, we found that BMI-1 expression was up-regulated in pancreatic cancer stem cells. Knockdown of BMI-1 expression attenuated invasion ability of pancreatic cancer stem cells in Transwell system and liver metastasis capacity in nude mice which were injected CSCs through the caudal vein. We are the first to reveal that BMI-1 could promote invasion and metastasis ability of pancreatic cancer stem cells. Finally, we identified that BMI-1 expression activating PI3K/AKT singing pathway by negative regulating PTEN was the main mechanism of promoting invasion and metastasis ability of pancreatic CSCs. In summary, our findings indicate that BMI-1 could be used as the therapeutic target to inhibiting CSCs-mediated pancreatic cancer metastasis.

Prognostic value of Notch-1 expression in hepatocellular carcinoma: a meta-analysis.

Association of Notch-1 expression with prognosis of patients with hepatocellular carcinoma (HCC) remains controversial. We conducted a meta-analysis to reevaluate the association of Notch-1 expression with clinicopathological characteristics and prognosis of HCC. PubMed, Embase, Web of Science, and China National Knowledge Infrastructure were searched to look for relevant studies. The association between Notch-1 expression and clinicopathological parameters and overall survival (OS) was then reassessed using the meta-analysis for odds ratio (OR) or hazard ratio (HR) and 95% confidence interval (CI). A total of seven studies, including 810 HCC patients, were eligible for the meta-analysis. Our data showed that high Notch-1 expression was able to predict poor OS (HR 1.50, 95% CI 1.17-1.83, P=0.0001). The pooled OR showed that high Notch-1 expression was significantly associated with tumor metastasis (OR 0.37, 95% CI 0.16-0.86, P=0.02) and tumor size >5 cm (OR 0.48, 95% CI 0.26-0.88, P=0.02). In contrast, there was no association between high Notch-1 expression and tumor differentiation, late TNM stage, tumor number, and portal vein invasion of HCC. In conclusion, Notch-1 overexpression might predict poorer survival and more aggressive behavior in patients with HCC.

[Role of oxidative stress and thioredoxin in gastric cancer].

OBJECTIVE: To explore the role of oxidative stress and the antioxidant protein thioredoxin in the tumorigenesis and progression of gastric cancer. METHODS: The plasma levels of adenosine deaminase (ADA), glutathione peroxidase (GPX), superoxide dismutase (SOD), and advanced oxidation protein products (AOPP) were determined by colorimetry, and the plasma levels of thioredoxin were determined by enzyme-linked immunosorbent assay (ELISA) in 48 gastric cancer patients and 30 healthy subjects. RT-PCR assay was employed to examine the expression levels of thioredoxin mRNA in the tissue samples of the patients. RESULTS: Compared with the healthy controls, patients with gastric cancer had significantly increased plasma levels of ADA and AOPP (P<0.05), decreased plasma GPX level (P<0.05), and similar plasma SOD levels. The plasma levels of thioredoxin were significantly higher in patients with gastric cancer than in the healthy controls (P<0.05). Thioredoxin levels was not associated with gender, age, degree of tumor cell differentiation, invasion depth, or lymph node metastasis (P>0.05), but was correlated to distant tumor metastasis (P<0.05). The expression of Trx mRNA was significantly higher in gastric carcinoma than in normal gastric tissue (P<0.05). CONCLUSION: Gastric cancer patients have high levels of oxidative stress and thioredoxin expression, and the latter is related to distant metastasis of the tumor.

Peptide-based immunotherapy for multiple myeloma: current approaches.

Multiple myeloma (MM) is a clonal B-cell malignancy with many fatal clinical sequelae. Despite extensive therapeutic approaches, cures remain rare exceptions. A recent promising area of investigation is the development of immunotherapeutic approaches that target and eliminate myeloma cells more selectively. Because of its potential to promote the destruction of cancerous cells via cytotoxic T-cell responses, peptide-based immunotherapy is one of these strategies to have attracted considerable attention. Furthermore, many studies were carried out to identify the best epitope peptides, the optimal vaccine formulation and schedule, and the preferable clinical situation for vaccination. Based on these results, various epitope peptides have been identified that may be selectively targeted by host immunity, and various approaches have been used to enhance the immune responses of peptides. This chapter focuses on reviewing previous immunotherapy trials, describing the current strategies for peptide-based immunotherapy, and discussing the achievable prospects in MM.

Involvement of oxidative stress in the relapse of acute myeloid leukemia.

The aims of the present study were to determine the level of oxidative stress and the salient factors leading to the relapse of acute myeloid leukemia (AML). Oxidative stress-related parameters and the expressions of specific genes were monitored in 102 cases of AML during a pretreatment period from a primary status to a relapse status. In addition, age-matched healthy subjects were classified as controls. The activities of adenosine deaminase and xanthine oxidase were higher in the relapse condition, whereas those of glutathione peroxidase, monoamine oxidase, and superoxide dismutase, and the total antioxidant capacity (T-AOC) were lower in the primary condition and in controls. Of particular note, levels of advanced oxidation protein products, malondialdehyde, and 8-hydroxydeoxyguanosine were also significantly higher in relapse patients. Furthermore, real-time PCR with SYBR Green revealed that the expression levels of human thioredoxin (TRX) and indoleamine 2,3-dioxygenase were increased in relapse patients. Pearson correlation analysis revealed that the T-AOC was positively correlated with GSH but negatively correlated with 8-OHdG, TRX, and indoleamine 2,3-dioxygenase. Linear regression showed that a low T-AOC and up-regulated TRX expression were the independent factors correlated with relapse. A strong association between oxidative stress and the incidence of disease relapse was observed, which has potential prognosis implications. These results indicate that oxidative stress is a crucial feature of AML and probably affects the development and relapse of AML.

Oxidative stress in depressive patients with gastric adenocarcinoma.

Recent data from several studies suggest that oxidative stress is involved in the biochemical mechanisms that underlie neuropsychiatric disorders. The present study was designed to investigate oxidative stress status in depressive patients with gastric adenocarcinoma (GA) at TNM stage III. Oxidative stress, depression and expression of specific genes were monitored during a pretreatment period. Serum total antioxidant capacity, catalase, superoxide dismutase concentrations, and antisuperoxide anion capacity (A-ASC) were significantly decreased in depressive patients compared to control subjects, whereas serum malondialdehyde (MDA) levels were significantly increased. Importantly, the formation of 8-hydroxy-deoxyguanosine (8-OHdG) accumulated. Furthermore, SYBR Green real-time PCR revealed that the expression levels of human oxoguanine glycosylase 1 and APEX nuclease 1 (APEX1) were increased in depressive patients. Pearson correlation analysis revealed that depression was positively correlated with SAS, SCL-90, MDA, 8-OHdG and APEX1, but negatively correlated with A-ASC. Thus, this study confirms oxidative imbalance in depressive patients with GA, and oxidative stress may play a role in the onset and exacerbation of depression.

The level of oxidative stress and the expression of genes involved in DNA-damage signaling pathways in depressive patients with colorectal carcinoma.

OBJECTIVES: This study investigated the connection among the oxidative stress, depression and expression of specific genes involved in DNA-damage signaling pathways in patients with colorectal carcinoma (CRC). METHODS: A unique Dukes'C subset of patients with newly diagnosed colorectal adenocarcinoma were assessed using the Hamilton Depression Rating Scale (HAMD), Zung Self-rating Depression Scale (SDS), Zung Self-rating Anxiety Scale (SAS), Symptom Checklist 90 (SCL-90) and other multiple-item questionnaires. Oxidative-stress-related parameters in sera and the expression of genes were monitored during a pretreatment period. RESULTS: Eighty-two eligibility cases were divided into 2 groups based on an HAMD score cutoff of 20: the mean score was 28.29 in Group A (depression, n=52) and 16.50 in Group B (nondepression, n=30). The serum total antioxidant capacity, catalase, and superoxide dismutase concentrations were lower in Group A, whereas those of nitric oxide and malondialdehyde were higher in Group A. Importantly, the 8-hydroxy-deoxyguanosine level was higher in Group A than in Group B (P<.05). Microarray analysis revealed that the expressions of p34, PA26, and ABL were higher in Group A, whereas those of HRAD51, CR6, and XRCC3 were higher in Group B. CONCLUSION: Oxidative stress is capable of causing neuronal toxicity via lipid peroxidation, DNA damage, and abnormalities of gene expression, and therefore is a possible pathogenic mechanism underlying depression in patients with CRC.

Expression and clinical significance of TAp73alpha, p53, PCNA and apoptosis in hepatocellular carcinoma.

AIM: To study the prognostic role of TAp73alpha, p53, proliferating cell nuclear antigen (PCNA) and apoptosis in patients with hepatocellular carcinoma (HCC) after surgical tumor ablation. METHODS: Forty-seven human resected HCC tissues and 42 adjacent non-cancerous tissues were studied with 10 normal liver tissues as control group. TAp73alpha, p53, and PCNA were detected with Elivision immunohistochemistry. Terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick-end labeling (TUNEL) method was used to detect the apoptosis cells. All clinical and pathological materials were analyzed by SPSS10.0 statistical package. RESULTS: TAp73alpha overexpressed in HCC tissues (36.2%) when compared with adjacent non-cancerous tissues (2.38%, P<0.005) and normal liver tissues (0, P<0.01). Mutant type p53 (mt-p53) overexpressed in HCC tissues (38.3%) when contracted with adjacent non-cancerous tissues (16.7%, P<0.05) and normal liver tissues (0, P<0.01). Proliferation index (PI) level in HCC tissues was significantly higher than that in adjacent non-cancerous tissues (30.34%+/-4.46% vs 27.88%+/-5.89%, t, P = 0.028). Apoptosis index (AI) level in HCC tissues was higher than that in adjacent non-cancerous tissues (8.62%+/-2.28% vs 7.38%+/-2.61%, t, P = 0.019). Expression of TAp73alpha was associated with lymph node metastasis and mt-p53, with r = 0.407 and 0.265, respectively. Expression of mt-p53 was associated with Edmondson's stage and AFP, with r = 0.295 and -0.357, respectively. In Kaplan-Meier univariant analysis, TAp73alpha, AFP, TNM stage, portal vein invasion, liver membrane invasion and HBsAg correlated with prognosis (log rank, P = 0.039, 0.012, 0.002, 0.000, 0.014, 0.007, respectively). Multivariant Cox regression analysis showed that TAp73alpha, AFP, TNM stage, portal vein invasion, liver membrane invasion and age were independent factors of prognosis. CONCLUSION: These results suggest that TAp73alpha can be used as a prognostic indicator of patients with HCC undergoing surgical tumor ablation. AFP, TNM, portal vein invasion, liver membrane invasion and age also have a potency of predicting the prognosis of HCC.

Impact of comorbid anxiety and depression on quality of life and cellular immunity changes in patients with digestive tract cancers.

AIM: A study was performed to investigate the impact of comorbid anxiety and depression (CAD) on quality of life (QOL) and cellular immunity changes in patients with digestive tract cancers. METHODS: One hundred and fifty-six cases of both sexes with cancers of the digestive tract admitted between March 2001 and February 2004 in the Department of Medical Oncology, First Affiliated Hospital of Xi'an Jiaotong University were randomly enrolled in the study. Depressive and anxiety disorder diagnoses were assessed by using the Structured Clinical Interview for DSM-IV. All adult patients were evaluated with the Hamilton depressive scale (HAMD, the 24-item version), the Hamilton anxiety scale (HAMA, a modified 14-item version), quality of life questionnaire-core 30 (QLQ-C30), social support rating scale (SSRS), simple coping style questionnaire (SCSQ), and other questionnaires, respectively. In terms of HAMD > or = 20 and HAMA > or = 14, the patients were categorized, including CAD (n = 31) in group A, anxiety disorder (n = 23) in group B, depressive disorder (n = 37) in group C, and non-disorder (n = 65) in group D. Immunological parameters such as T-lymphocyte subsets and natural killer (NK) cell activities in peripheral blood were determined and compared among the four groups. RESULTS: The incidence of CAD was 21.15% in patients with digestive tract cancers. The average scores of social support was 43.67+/-7.05 for 156 cases, active coping 20.34+/-7.33, and passive coping 9.55+/-5.51. Compared with group D, subjective support was enhanced slightly in group A, but social support, objective support, and utilization of support reduced, especially utilization of support with significance (6.16 vs 7.80, P<0.05); total scores of active coping decreased, while passive coping reversed; granulocytes proliferated, monocytes declined, and lymphocytes declined significantly (32.87 vs 34.00, P<0.05); moreover, the percentage of CD3, CD4, CD8 and CD56 in T lymphocyte subsets was in lower level, respectively, and CD56 showed a significant decline in group A (26.02 vs 32.20, P<0.05), however, CD4/CD8 ratio increased. Physical function, role function, fatigue, sleeplessness and constipation had significant changes among different groups by one-way ANOVA, and group A was in poor QOL. It revealed that global health-related quality of life (QL) were positively correlated with active coping and CD56; CAD was negatively correlated with QL, active coping and CD56. Furthermore, the step-wise regression analysis suggested that utilization of support, CD56, active coping, fatigue, sleeplessness and depression were significant factors contributing to QOL. CONCLUSION: CAD, which can impair QOL and cellular immunity, occurs with a higher incidence in patients with digestive tract cancers. Hence, it is essential to improve mental health for them with specifically tailored interventions.

Effects of depression on parameters of cell-mediated immunity in patients with digestive tract cancers.

AIM: To evaluate the effects of depression on parameters of cell-mediated immunity in patients with cancers of the digestive tract. METHODS: One hundred and eight adult patients of both sexes with cancers of the digestive tract admitted between March 2001 and February 2002 in the Department of Medical Oncology, First Affiliated Hospital of Xi'an Jiaotong University were randomly enrolled in the study. The Zung self-rating depression scale (SDS), Zung self-rating anxiety scale (SAS), numeric rating scale (NRS) and social support rating scale (SSRS) were employed to evaluate the degree of depression and their contributing factors. In terms of their SDS index scores, the patients were categorized into depression group (SDS> or =50) and non-depression group (SDS<50). Immunological parameters such as T-lymphocyte subsets and natural killer (NK) cell activities in peripheral blood were determined and compared between the two groups of patients. RESULTS: The SDS index was from 33.8 to 66.2 in the 108 cases, 50% of these patients had a SDS index more than 50. Similarly, the SAS index of all the patients ranged from 35.0 to 62.0 and 46.3% of the cases had a SAS index above 50. Cubic curve estimation showed that the depression was positively correlated with anxiety and negatively with social support. Furthermore, the depression correlated with the tumor type, which manifested in a descending order as stomach, gallbladder, pancreas, intestine, esophagus, duodenum and rectum, according to their correlativity. Step-wise regression analysis suggested that hyposexuality, dispiritment, agitation, palpitation, low CD56 and anxiety were the significant factors contributing to depression. More severe anxiety (49.7 +/- 7.5 vs 45.3 +/- 6.9, P<0.05), pain (6.5 +/- 2.8 vs 4.6 +/- 3.2, P<0.05), poor social support (6.8 +/- 2.0 vs 7.6 +/- 2.1, P<0.05), as well as decline of lymphocyte count (0.33 +/- 0.09 vs 0.39 +/- 0.87, P<0.05) and CD56 (0.26 +/- 0.11 vs 0.29 +/- 0.11, P<0.05) were noted in the depression group compared with those of the non-depression patients. However, fewer obvious changes in CD4/CD8 ratio and other immunological parameters were found between the two groups. CONCLUSION: Depression occurs with a high incidence in patients with cancers of the digestive tract, which probably is not the sole factor leading to the impairment of immunological functions in these cases. However, comprehensive measures including psychological support should be taken in order to improve the immunological function, quality of life and clinical prognosis of these patients.

Cloning of human 15ku selenoprotein gene from H9 T cells.

AIM: To clone human 15ku selenoprotein gene. METHODS: H9 human T cells were cultured in RPMI1640 medium supplemented with 100 mL /L fetal calf serum. mRNA was isolated from the cells. cDNA library was constructed by RT-PCR. The human 15ku selenoprotein gene was obtained by PCR and cloned into T vector and sequenced. RESULTS: A unique cDNA fragment about 1 244 bp was obtained. Sequence analysis identified an open reading frame within the cDNA. The gene had an in-frame TGA, which encoded selenocysteine (Sec), and a 3'-UTR SECIS element, which was required for synthesis of selenoprotein. The predicted protein molecular mass was about 15ku (162 residues). The result was identical with human liver 15ku selenoprotein gene published in Genbank. CONCLUSION: Human 15ku selenoprotein gene can be successfully obtained from T cell line.