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CANT1 serves as a potential prognostic factor for lung adenocarcinoma and promotes cell proliferation and invasion in vitro
BMC Cancer volume 22, Article number: 117 (2022)
Calcium-activated nucleotidase 1 (CANT1), functions as a calcium-dependent nucleotidase with a preference for UDP. However, the potential clinical value of CANT1 in lung adenocarcinoma (LA) has not been fully clarified. Thus, we sought to identify its potential biological function and mechanism through bioinformatics analysis and in vitro experiments in LA.
In the present study, we comprehensively investigated the prognostic role of CANT1 in LA patients through bioinformatics analysis and in vitro experiments. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were utilized to analyze the expression of CANT1 in LA patients and their clinical-prognostic value. The immunohistochemistry staining was obtained from the Human Protein Atlas (HPA). A Cox regression model was used to evaluate prognostic factors. Gene ontology (GO) and Gene set enrichment analysis (GSEA) was performed to explore the potential regulatory mechanism of CANT1 in the development of LA. Moreover, we also examined the relationship between CANT1 expression and DNA methylation. Finally, we did in vitro experiments to evaluate the biological behavior and role of CANT1 in LA cells (LACs).
Our study showed that the CANT1 expression was significantly elevated in the LA tissues compared with the normal lung tissues. Increased CANT1 expression was significantly associated with the TN stage. A univariate Cox analysis indicated that high CANT1 expression levels were correlated with poor overall survival (OS) in LA. Besides, CANT1 expression was independently associated with OS in multivariate analysis. GO and GSEA analysis showed the enrichment of mitotic nuclear division, DNA methylation, and DNA damage. Then we found that the high expression of CANT1 is positively correlated with hypomethylation. The methylation level was associated with prognosis in LA patients. Finally, in vitro experiments indicated that knockdown of CANT1 resulted in decreased cell proliferation, invasion, and G1 phase cell-cycle arrest in LACs.
The present study suggested that CANT1 may serve as a potential prognosis biomarker in patients with LA. High CANT1 expression and promoter demethylation was associated with worse outcome. Finally, in vitro experiments verified the biological functions and behaviors of CANT1 in LA.
Lung cancer is the leading cause of cancer death and the second most common cancer in both men and women . Among them, the incidence of lung adenocarcinoma (LA), the most common histological type of lung cancer, is increasing year by year [2,3,4]. Despite immense progress in therapeutic strategies, including surgery, molecular targeted agents, and immune checkpoint inhibitors, the 5-year survival rate among patients with non-small cell lung cancer (NSCLC) is less than 20% [5,6,7]. The high mortality for NSCLC patients may be driven by two factors: one is that about 66% of NSCLC patients are diagnosed at a relatively advanced stage. The other one is that even in the early-stage portion, the mortality rate is still very high because of the high recurrence rate after surgery [8, 9]. Therefore, there is an urgent need to identify and develop novel molecular markers to improve the prognosis of LA.
Calcium-activated nucleotidase 1 (CANT1), also known as DBQD, SCAN-1, SCAN1, SHAPY, functions as a calcium-dependent nucleotidase with a preference for uridine diphosphate (UDP). The protein encoded by CANT1 belongs to the apyrase family. CANT1 is generally expressed in human tissues . Alternative splicing transcript variants have been noted for this gene. The alternative splicing transcript variants often appear in cancer and have been regarded as an important sign of tumor progression and cancer treatment . Cancer cells often show abnormal alternative splicing profiles to produce isoforms, stimulating cell proliferation and migration or increasing apoptosis resistance . CANT1 is a new oncogenic mRNA in some cancer types, like human clear cell renal cell carcinoma (ccRCC) and prostate cancer. These studies showed that CANT1 was associated with cancer cell proliferation, migration, and invasion. Besides, the high expression of CANT1 was positively correlated with TNM stage and histological grade in ccRCC [13, 14]. In addition, some studies have indicated that mutations in CANT1 are associated with abnormal growth and development [15, 16]. Previous research has shown that factors related to normal growth often play a momentous role in tumor growth . These results suggest the potential role of CANT1 as a prognostic biomarker in cancer. Although CANT1 plays a crucial role in some types of tumors, it is unclear whether it is a prognostic factor of LA. Besides, there is little literature about the relationship between CANT1 and LA reported so far. It is the rare study to demonstrate the functional mechanism of CANT1 in LA.
CANT1 is a phosphatase whose preferred substrate is UDP and followed by GDP, UTP, GTP, ADP, and ATP . Gerhardt et al. indicated a G1 phase cell-cycle arrest on CANT1 knockdown in LNCaP and PC-3 cells . Another result showed that knockdown of CANT1 induced an S-phase cell-cycle arrest, resulting in a decrease in the number of cells entering the G2/M-phase . It also demonstrated that CANT1 knockdown could promote apoptosis. Several genes involved in the apoptotic pathways were altered after CANT1 was knocked out in 769-P cells. Such as the pro-apoptotic factors of BAD and cleaved caspase-3 were upregulated, while the anti-apoptotic factor of BCL2 was downregulated. Serine, glycine, threonine metabolism, purine metabolism, and glycosphingolipid biosynthesis in the genes’ pathway are significantly altered . Besides, CANT1 might contribute to tumorigenesis via activating both MAPK and NF-kB signaling pathways . However, little literature has explored the association between CANT1 and LA. Therefore, our study aimed to elucidate the expression of CANT1 and its potential value of therapy and prognosis in LA tissues.
In the present study, we comprehensively investigated the prognostic role of CANT1 in LA patients through bioinformatics analysis and in vitro experiments. This research aimed to identify CANT1 gene with potential prognostic relevance in patients with LA. Up to now, there is no evidence that CANT1 relates to the occurrence and development of LA. Therefore, our study aimed to elucidate the expression of CANT1 and its potential value of therapy and prognosis in LA tissues. Besides, there is little literature about the relationship between CANT1 and LA reported so far. It is the rare study to demonstrate the functional mechanism of CANT1 in LA. In the study, we explored the effect of CANT1 expression and methylation on prognosis to determine whether CANT1 can be a potential prognostic biomarker for patients with LA. Finally, we also verified the biological function and behavior of CANT1 in LACs through in vitro experiments. This further strengthens our understanding of the biological mechanism of CANT1.
Clinical information from TCGA and GEO databases
The lung adenocarcinoma tissues and adjacent tissues included in our study were from the TCGA database, where a total of 502 LA patients from 1991 to 2013. Gene expression data and the corresponding clinical information of the TCGA-LA were obtained from the TCGA (https://portal.gdc.cancer.gov/). The GEO databases of GPL570 and GPL6884 platforms were performed for the validation (www.ncbi.nlm.nih.gov/geo). Expression profiles (HTSeq-Counts) were compared between high and low CANT1 expression groups to identify differentially expressed genes (DEGs) using the DESeq2 R package. The data used in this study were in accordance with the guidelines provided by TCGA and GEO. The ethics approval and informed consent were not required.
Over-expression of CANT1 in LA patients
We explored the expression differences of CANT1 in LA patients between tumor tissue and normal tissue. Besides, the immunohistochemistry staining of CANT1 expression was obtained by the Human Protein Atlas (HPA) (https://www.proteinatlas.org) [19, 20]. On the HPA website, antibodies HPA019627, HPA022818, and HPA019639 were used for immunohistochemistry staining of CANT1.
Role of CANT1 in LA patient’s survival
A total of 502 LA patients were included in our study. The survival curve was performed by survival R package and survminer R package. Furthermore, we validated the prognostic values of the risk model in two independent datasets from GSE31219 (n = 83) and GSE41271 (n = 181).
Construction and evaluation of the nomogram model
A nomogram was built based on the multivariate analysis’s result to predict the survival probability for 1-year, 3-years, and 5-years of LA patients. The rms R package was performed to produce a nomogram. The concordance index (C-index) and calibration curve were performed by the Hmisc R package. In this study, C-index was carried out to determine the nomogram’s discrimination with 1000 bootstrap replicated.
Functional enrichment analysis
GEPIA is a database based on the UCSC Xena project . In the study, GEPIA 2.0 (http://gepia2.cancer-pku.cn/) was performed to find the gene with the most similar expression of CANT1 in LA. Finally, 50 most correlated co-expression genes were founded. Then the functional analysis of CANT1 and its co-expression genes in TCGA-LA were predicted by Metascape (http://metascape.org) .
Gene set enrichment analysis
Gene set enrichment analysis (GSEA) is a calculation method that determines whether a set of prior defined genes show statistically significant and consistent differences between two biological states [23, 24]. In this study, GSEA was performed using the Molecular Signatures Database (MSigDB) Collection (c2.all.v7.0.entrez.gmt) of clusterProfiler R package to illuminate the statistically significant pathway difference between high and low CANT1 expression groups of LA. The expression level of CANT1 was used as a phenotype label. The pathway terms with adjusted P-value < 0.05 and false discovery rate (FDR) q-value < 0.25 were considered significantly enriched.
Correlation between CANT1 expression and DNA methylation
The previous GSEA analysis suggested that CANT1 was related to DNA methylation. Therefore, we then analyzed the relationship between the expression of CANT1 and DNA methylation. Spearman correlation coefficient was performed to reveal the correlation between the expression of CANT1 and methylation level in TCGA-LA. The gene methylation data of the TCGA-LA project was obtained from the UCSC Xena browser (version:07-20-2019, https://xenabrowser.net/datapages/). The relationship between the methylation level and overall survival (OS) of CANT1 was retrieved from MethSurv (https://biit.cs.ut.ee/methsurv/). It is a web tool to provide univariable and multivariable survival analysis based on DNA methylation biomarkers by using TCGA data .
Cell lines and cell culture
Human lung adenocarcinoma cell lines A549 and H1299 were obtained from the Genechem company (Shanghai, China). A549 cells were cultured in Ham’s F-12 K medium (PYG0036, Boster, USA) supplemented with 10% fetal bovine serum (FBS, P30-3302, PAN biotech, Germany) and 1% penicillin/streptomycin (100x, Gibco, USA), H1299 Cells were cultured in RPMI-1640 (PYG0006, Boster, USA) supplemented with 10% FBS and 1% penicillin/streptomycin. All cell lines were maintained in a humidified incubator with 5% CO2 at 37 °C. The medium was changed every 3 days, and a subcultivation ratio of 1:3 was performed.
Small interfering RNAs transfection
Small interfering RNAs (siRNAs) were used to selectively knockdown the expression of CANT1 by the Lipofectamine 3000 transfection reagent (L3000-015, Invitrogen, USA), according to the manufacturer’s instructions. The sequence of three groups of siRNAs targeting CANT1 (siRNA-987, siRNA-1173, and siRNA-273) and two negative control groups (si-NC and NC) were obtained from the zolgene (Fuzhou, China) shown in Table 1. For transfection, A549 and H1299 cells in the logarithmic growth phase were inoculated into a six-well plate. Knockdown efficiency was verified on the RNA level using quantitative real-time polymerase chain reaction (qRT-PCR).
Quantitative real-time polymerase chain reaction
Total RNA was isolated using the TRIzol™ Reagent (15,596,026, Invitrogen, USA). Then the GoScript™ Reverse Transcription System (A5001, Promega, USA) was employed to reversely transcribe RNA into cDNA. qRT-PCR was conducted using UltraSYBR Mixture (CW0957, Cwbio, China) to measure the expression of CANT1. β-actin was used as the internal reference. The calculation of inhibitory efficacy of the CANT1 mRNA was calculated using the 2-ΔΔCt method. The sequences of primers used in qRT-PCR were shown in Table 2.
Cell counting kit-8 (CCK-8) assay
After transfection, we used the CCK-8 reagent (40203ES60, Yeasen, Shanghai, China) to measure cell viability. The si-CANT1 group, si-NC group, and NC group of A549 and H1299 cells were subcultured in a 96-well plate and then incubated for 0, 24, 48, and 72 h. Before detection, 10 μl (10%) CCK8 reagent was added daily to each hole in the 96-well plates and incubated at 37 °C for 1 h. Then, a microplate reader (Nano-100, Allsheng, Hangzhou, China) was used to measure the absorbance at 450 nm.
Cell invasion assay
The invasive ability of the A549 and H1299 cells was assessed with Transwell chambers and Matrigel. The Transwell chambers were coated with 50 μl diluted Matrigel (1:8) at 37 °C for 4 h. Subsequently, 1 × 105/mL cells resuspended in serum-free medium were added into the Transwell chamber, which was placed in cell culture medium supplemented with 10% FBS and was cultured for 48 h. Then the residual cells in the upper chamber were wiped with cotton swabs. Cells migrated through the membrane were fixed with 4% paraformaldehyde (P1110, Solarbio, Beijing, China), washed with PBS, mixed with 0.4% crystal violet solution (C0121, Beyotime, Shanghai, China), and counting in 3 randomly picked view under a microscope at 100× magnification (MF53, Mshot, Guangzhou, China).
For cell cycle detection, 1 × 106/ml cells were counted and washed with phosphate buffer saline (PBS, ABA212278, Hyclone, USA), followed by the addition of 70% cold ethanol at 4 °C overnight. After washing with cold PBS, the fixed-cell pellets were collected by centrifugation and resuspended in ribonuclease (RNase) A/Propidium Iodide (PI) staining buffer. The cell-cycle distribution was analyzed by flow cytometer (FACSVerse, BD, USA).
All statistical analysis was performed using SPSS (version 26.0) and R (version 4.0.2). Wilcoxon rank-sum test and Wilcoxon signed-rank test were used to compare the expression of CANT1 between LA and normal groups. X-tile software (version 3.6.1, https://medicine.yale.edu/lab/rimm/research/software/) was performed to seek the optimal cutoff value of CANT1 expression. The relationship between the TN stage and the expression of CANT1 was analyzed with Wilcoxon single-rank test. Clinical parameters (age, gender, number pack years smoked, epithelial growth factor receptor (EGFR) mutation status, kirsten rat sarcoma viral oncogene (KRAS) mutation status, anaplastic lymphoma kinase (ALK) mutation status, T stage, N stage, M stage, pathological stage, and gene expression) associated with OS in LA patients were evaluated with Cox analyses. In univariate analysis, all factors with P < 0.20 were involved in multivariate analysis to identify independent prognostic factors. Owing to missing data exceeding 20%, the ALK mutation status and M stage were not included in multivariate analysis. Comparisons between two groups were analyzed by t-test. All experiments were performed with at least three independent replications. All tests were two-sided, and a P-value < 0.05 was considered statistically significant.
Clinical information from TCGA database
The clinical data including 502 patients were collected from TCGA (Table 3).
Over-expression of CANT1 in LA patients
We used the Wilcoxon rank-sum test to investigate the expression of CANT1 in 502 LA tissues and 57 normal tissues. The expression of CANT1 was significantly higher in LA groups than in normal groups (P < 0.001) (Fig. 1A). In addition, we further used Wilcoxon singed-rank test to examine the expression of CANT1 in 57 pairs of LA tissues. The result showed CANT1 was significantly overexpressed in LA (P < 0.001) (Fig. 1B). As shown in Fig. 1C-D, increased CANT1 expression in LA was significantly associated with the TN stage (T1 vs. T2/T3/T4, P = 0.042; NO/ N1 vs. N2/N3, P = 0.041). We also carried out an analysis of the association between CANT1 expression and M (metastasis) status. Due to the result are negative and meaningless, we did not add this result to the Figure. These results demonstrated that the CANT1 expression level could affect LA patients’ prognosis with different TN stage. After exploring the CANT1 expression patterns of CANT1 in LA, we tried to examine the protein expression patterns of CANT1 in LA by the HPA. As shown in Fig. 1E-J, low protein expressions of CANT1 were observed in normal lung tissues, while high protein expressions were detected in LA tissues. The staining intensity was scored as negative, weak and negative in HPA019627, HPA022818, and HPA019639 in normal lung tissues. While the staining intensity was scored as Strong in HPA019627, HPA022818, and HPA019639 in LA tissues. The quantity was scored as none, < 25%, and none in HPA019627, HPA022818, and HPA019639 in normal lung tissues. While the quantity was scored as > 75, > 75, and 25%-75% in HPA019627, HPA022818, and HPA019639 in LA tissues.
Role of CANT1 in LA patient’s survival
Overall survival (OS) was significantly reduced in patients with high CANT1 expression than those with low CANT1 expression (P = 0.005) (Fig. 2A). To validate the association between the expression of CANT1 and OS, we also performed the GSE30219 and GSE41271 datasets from the GEO. The outcome indicated that OS was significantly reduced in patients with high CANT1 expression than those with low CANT1 expression (P = 0.018, P = 0.043, respectively) based on GSE30219 and GSE41271 database (Fig. 2B-C). We further used the univariate Cox regression model to identify prognostic factors in LA (Table 4). The result showed that high CANT1 expression levels were correlated with worse OS (P = 0.005, hazard ratio [HR] = 1.547 (95% confidence interval [CI] [1.143-2.094])). In addition, age > 70 years old (P = 0.013, HR = 1.464(95% CI [1.083-1.979]), higher TNM stage (T: P = 0.004, HR = 1.666 (95%CI [1.182-2.347]); N: P < 0.001, HR = 2.265 (95% CI [1.584-3.239]); M:P = 0.006, HR = 2.143 (95% CI [1.251-3.672])), and higher pathological stage (P < 0.001, HR = 2.609 (95%CI [1.911-3.561])) were also associated with poor OS. We then performed a multivariate analysis with the Cox regression model. Due to the missing data of ALK mutation status and M stage over 20%, they were not included in the multivariate analysis. The result indicated that CANT1 expression (P = 0.015, HR = 1.490 (95%CI [1.081-2.055])), T stage (P = 0.032, HR = 1.467(95% CI [1.034-2.081])) and pathological stage (P = 0.001, HR = 2.290 (95% CI [1.395-3.758])) were independently associated with worse OS.
Development of a prognostic model based on the expression of CANT1 and clinical factors
A nomogram integrating the expression of CANT1, T stage, and pathological stage was constructed in our study (Fig. 3A). A worse prognosis was represented by a higher total number of points on the nomogram. The C-index was 0.64 based on 1000 bootstrap replicates for the nomogram. The deviation correction line in the calibration plot was close to the ideal curve, showing that the prediction result is in good agreement with the observation results (Fig. 3B-D).
CANT1-related function enrichment obtained by Metascape
The GO enrichment items were engaged in several functional groups (Fig. 4A-C): cellular component (8 items), biological process (16 items), and molecular function (8 items). The cellular components for CANT1 and its most similar genes were mainly involved in cell-cell junction, cell cortex, and cell body. The biological processes were enriched in cytoskeleton-dependent cytokinesis, neutrophil-mediated immunity, Ras protein signal transduction, apoptotic signaling pathway, and mitotic nuclear division. The molecular functions were mainly engaged in GDP binding and Ras GTPase binding. Figure 5A showed that an interactive network of the top 19 enrichment terms. It is colored by cluster-ID. Distinct colors are various enrichment pathways of CANT1 correlated genes.
CANT1-related signaling pathways obtained by GSEA
The TCGA RNA-seq data was used to compare CANT1-high and CANT1-low groups’ gene expression in LA. Finally, there are 19,479 differentially expressed genes were identified between the CANT1-high group and CANT1-low group. In order to gain further insight into the biologic pathways involved in LA by the expression level of CANT1, all the differentially expressed genes were analyzed by GSEA. The significantly enriched signaling pathways, mainly including depurination, DNA methylation, and DNA damage telomere stress-induced senescence are enriched based on normalized enrichment score (NES), adjusted P-value, and FDR value (Fig. 5B).
Correlation between CANT1 expression and methylation
Figure 6A showed that the methylation level was significantly negatively correlated with CANT1 expression with Spearman up to − 0.2561 (P < 0.001). The effect of hypomethylation level and the expression of CANT1 on prognosis in LA was obtained from MethSurv. We discovered that 3 CpG sites located on the CpG island indicated a poor prognosis, including cg00902147, cg16337457, and cg18186771 (Fig. 6B-D).
The expression levels of CANT1 in LAC lines
We constructed A549 and H1299 cells by endogenously knocking down CANT1 by transfection of specific siRNAs, named as A549 si-CANT1 and H1299 si-CANT1. The expression of CANT1 was silenced by three siRNAs, as result, the qRT-PCR assay showed that si-1173 and si-273 had the best silencing effect among the three siRNAs against CANT1 in A549 and H1299 cell lines, respectively (Fig. 7A-B). The results indicated that si-1173 and si-273 were the most effective to inhibit the expression of CANT1 in A549 and H1299 cell lines, respectively. Therefore, they were selected in subsequent experiments.
Cell counting kit-8 (CCK-8) and cell invasion experiments
To explore the effects of CANT1 on LACs’ proliferation and invasion, a series of in vitro experiments were carried out. The results of CCK-8 assays showed significant decline in the proliferation of A549 and H1299 cells after the knockdown of CANT1 (P < 0.001) (Fig. 7C-D). Transwell assay was conducted to evaluate the effects of CANT1 on cell invasion. CANT1 knockdown in A549 and H1299 cells remarkably inhibited the invasion ability (P < 0.001) (Fig. 8).
Flow cytometry experiment
To better understand the mechanisms of cell proliferation inhibited, the percentages of cells in different phases of the cell cycle were analyzed by flow cytometry. A significant increase in the G1 phase was observed in the si-CANT1 group (69.35 and 82.16%), compared with the NC group and si-NC group (62.41 and 69.65%, 62.43% and 71.66, respectively) in A549 and H1299 cells. Cell cycle was arrest in the G1 phase in the Si-CANT1 group, resulting in less cells entering the S phase (Fig. 9). Summarized, these results confirmed that knockdown of CANT1 could significantly inhibit the malignant phenotypes of LACs.
Lung adenocarcinoma (LA) accounts for almost half of lung cancers and patients with advanced LA have a very low survival rate . Despite advances and efforts in understanding LA biology in recent years, the functional mechanisms of LA carcinogenesis remain elusive. This research aimed to identify gene with potential prognostic relevance. Up to now, there is no evidence that CANT1 relates to the occurrence and development of LA. Nevertheless, the role of CANT1 has been confirmed in other cancers, including clear cell renal cell carcinoma (ccRCC)  and prostate cancer . To the best of our knowledge, it is the rare study to demonstrate the functional impact of CANT1 in LA.
Several studies have reported the prognostic significance of CANT1 for some cancers. High CANT1 expression was related to having a poor overall survival (OS) rate (P < 0.05) in ccRCC .The prognostic analysis was also carried out in our study. High expression of CANT1 was correlated with poor prognosis of LA in T2 to T4, N1 to N3, and stage II to IV. As shown above, our study suggested that CANT1 is a prognostic biomarker in LA. High expression of CANT1 indicated a worse survival rate.
In this study, GO enrichment analysis including Ras protein signal transduction, Ras GTPase binding, mitotic nuclear division, apoptotic signaling pathway, and cytoskeleton-dependent cytokinesis. These results suggested that CANT1 is related to cell division and growth, further supporting its relationship with tumorigenesis. Other enrichment results are related to intercellular recognition, interaction, and metabolism, which need to be confirmed by further study. GSEA analysis including DNA methylation, DNA damage telomere stress-induced senescence, and depurination. Evan et al. indicated that excessive accumulation of cells in cancer cells results from excessive cell proliferation and inadequate apoptosis. The inactivation of pro-apoptotic genes and the increased expression or activity of anti-apoptotic proteins lead to apoptosis deficiency . Depurination is a process in which purines are lost in nucleic acids due to energy absorption. It participated in maintaining cell metabolism, which may affect cell growth. DNA depurination was related to the occurrence and development of cancer . The previous study had also shown that CANT1 was involved in the therapy of purine and pyrimidine antimetabolites in cancer .
Several studies have demonstrated that the initiation and progression of tumors can be facilitated by epigenetic and genomic alterations , such as DNA methylation . Despite many mechanisms contributing to elevated gene expression, DNA methylation is one of the most common mechanisms. Sato et al. indicated that high gene expression induced by hypomethylation was associated with poor prognosis in NSCLC [32, 33]. Through our analysis, we found that hypomethylation is correlated with the high expression level of CANT1. Our studies also showed that the hypomethylation level was associated with poor prognosis in LA patients. Therefore, CANT1 expression may be epigenetically regulated by DNA hypomethylation and has prognostic significance for LA. This result suggested that the hypomethylation of CANT1 may be associated with the high expression of CANT1.
To further confirm the biological mechanism of CANT1, we carried out a series of in vitro experiments in A549 and H1299 cells. Our experimental results indicated that knockdown of CANT1 resulted in decreased cell proliferation, invasion, and G1 phase cell-cycle arrest in LACs, which is consistent with our bioinformatics prediction. Based on the above results, CANT1 may take an active part in cell growth and division in LA and leading to the occurrence and development of LA.
Although the present study enhanced our understanding of the relationship between CANT1 and LA, our study had some limitations. First, our research was validated by in vitro experiments, we did not perform relevant experiments in vivo. Additionally, our investigations into the role of CANT1 in the tumor were based on the TCGA and GEO databases, which lacks verification from our clinical samples. Finally, we also cannot clearly investigate the direct mechanisms of CANT1 involved in the occurrence and development of LA. Our research has indicated the role of CANT1 in the metastasis of LA cells. Herein the results have shown that the CANT1 plays an important role in the invasion and migration of LA cells. It showed that the overexpression of CANT1 caused significant increase in the invasion and migration of LA cells, indicative of the role of CANT1 in LA cell metastasis. Although both CANT1 siRNA have interference effects, we have verified the most effective one. Nonetheless, it is fairly important to use one more CANT1 siRNA for the important experiments. At present, our research is mainly based on bioinformatics analysis and preliminary cell experiments to explore the role of CANT1 in LA, so in our future plan, we will further explore CANT1-mediated downstream pathways or molecules, so as to clarify the specific mechanism of CANT1 affecting the malignant phenotype of LA. It might be interesting to study whether the CANT1 gene may affect the biological function and mechanism of LA. Anyhow, a large number of samples and comprehensive studies are still required to strengthen the hypothesis that CANT1 is a prognostic biomarker.
In conclusion, the present study suggested that CANT1 may serve as a potential prognostic biomarker in patients with LA. High CANT1 expression and promoter demethylation was associated with worse outcome. Finally, in vitro experiments verified the biological functions and behaviors of CANT1 in LACs. The specific mechanisms about knockdown of CANT1 expression inhibited the growth of LACs still need further research.
Availability of data and materials
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Calcium-activated nucleotidase 1
The Cancer Genome Atlas
Gene Expression Omnibus
Non-small cell lung cancer
Lung adenocarcinoma cells
Gene set enrichment analysis
Human clear cell renal cell carcinoma
Differentially expressed genes
Human protein atlas
Molecular signatures database
Single-sample gene set enrichment analysis
False discovery rate
Normalized enrichment score
Fetal bovine serum
Small interfering RNAs
Quantitative real-time polymerase chain reaction
Cell counting kit-8
Phosphate buffer saline
Epithelial growth factor receptor
Kirsten rat sarcoma viral oncogene
Anaplastic lymphoma kinase
Regional lymph node
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We sincerely thank the public databases, including TCGA, GEO, GEPIA, Metascape, HPA, and MethSurv for providing open access.
This work was supported by the grants of the Wu Jieping Foundation (320.6750.19053), the National Natural Science Foundation of China (81803037), and Joint Funds for the innovation of science and Technology, Fujian province (2018Y9111, 2019Y9041).
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Yao, Q., Yu, Y., Wang, Z. et al. CANT1 serves as a potential prognostic factor for lung adenocarcinoma and promotes cell proliferation and invasion in vitro. BMC Cancer 22, 117 (2022). https://doi.org/10.1186/s12885-022-09175-2
- Lung adenocarcinoma

domain: biology


text: Download or read book If Not Now When written by Ronald P. Culberson and published by Greenleaf Book Group. This book was released on 2017-01-10 with total page 152 pages. Available in PDF, EPUB and Kindle. Book excerpt: The simple question about becoming a better person is If Not Now, When? In his previous book, Do it Well. Make it Fun, author Ron Culberson introduced us to the idea that excellence coupled with fun and humor can help us achieve both success and contentment in all areas of life and work. Culberson draws on his experience as a hospice social worker and his expertise in the benefits of humor and laughter to further this work in his new book, a collection of blog posts, articles, and essays. In If Not Now, When? Making the Most of Your Life, Your Relationships, and Your Work, Ron offers surprising wisdom into these three areas of life, couched in entertaining anecdotes and self-effacing humor. Ron tackles every aspect of life, from how to make your job more enjoyable to some of our most difficult challenges, like parenting, aging, and death. Readers will learn how to be present in the moment, embrace a commitment to empathy, and truly notice the world around them in ways that they may never have. And all along the way, they will enjoy the journey through the author’s amusing perspective.

domain: literature


text:
ITEM PHOTO | SPENSER HASAK
Nathaniel Parker of Portsmouth, N.H., reacts to the sale of Whole Foods to Amazon.
By MATT DEMIRS
LYNNFIELD — Whole Foods Market customers didn’t have anything bad to say after the Internet giant, Amazon, purchased the grocery store Friday morning.
They did raise one concern, however. They don’t want change.
“I’ll keep shopping here,” said Nathaniel Parker of Portsmouth, New Hampshire, “as long as they don’t mess up the formula.”
Amazon purchased the grocery chain at $42 a share, or $13.4 billion, making it the largest deal in company history. Amazon’s CEO Jeff Bezos is reportedly paying a 27% premium on Whole Foods shares.
Whole Foods, which opened its first location on September 20, 1980 in Austin, Texas, has 30 locations in Massachusetts, according to their website.
The purchase raises shoppers’ concerns as to what might change in their beloved Lynnfield and Swampscott locations.
Carol Herdic, who is visiting her daughter from Miami, wasn’t aware of the purchase but is very familiar with Amazon and its services.
“I order from it almost every day,” she said. “I own Echo and Alexa and buy certain groceries on there, although I don’t use Amazon Pantry. I do, however, use the subscribe and save feature.”
She too hopes Amazon doesn’t have any plans to change Whole Food’s current product.
“I find it very interesting because I heard Amazon cuts its price in order to get to its customer,” she said. “And you don’t think of that when you think of Whole Foods since it is more of a premium market.
Although Herdic said she’s unsure of what might become of the two companies, it might mean that Amazon is changing its business model, or that Whole Foods might becoming cheaper, she said. She’s interested to see what will happen.
The news comes after Amazon expanded into the brick and mortar industry earlier this year as the Internet legend opened eight bookstores across the country, with more set to come.
The store manager at Amazon Books in Lynnfield declined to comment on the purchase of Whole Foods, citing restrictions placed on employees regarding talking with the media.
The manager of Whole Foods in Lynnfield was not available for a comment at this time.
Emily Brengle of Ipswich said she doesn’t really have any idea of what might become of the store as she loaded her car with groceries at the Lynnfield location.
She uses Amazon for online shopping and really enjoys Amazon Prime, the company’s two-day shipping service.
“The only thing I care about is whether Whole Foods is going to change,” she said.
Matt Demirs can be reached at email@example.com

domain: economics


text:
Super Retro World : Pixel Art Maker（MOD (Unlimited Coins) v220.127.116.11）
- Current Version:
- File size: 2G
1、The gold standard has returned to the open-world action game, this time in the fascinating city of New Orleans. With hundreds of vehicles, an insulting arsenal, explosive action, and complete freedom to explore this huge city, you have all the tools to become a real Gangster.
Around three weeks ago Niantic announced that would be introducing a new feature to Harry Potter: Wizards Unite that would allow players to grow their magical skills. Now we know exactly what that is, wizards and witches that are level 4 and above will get access to a new set of SOS Training skill trees.

This will give players access to a host of lessons that will unlock a plethora of bonuses and rewards. The SOS Task Force is set to roll-out in the next few days and will allow players to improve abilities such as spell-casting, potion-brewing and charm-casting.

Among those new skills will be Mastery. This will buff the power of your spells, which should help when dealing with Confoundables. Alternatively, if you frequently find that Foundables are escaping you then you might want to go with Departure Denial which reduces the chances of this happening.
The Academy The First Riddle review - "Unproblematic Potter with some caveats"
The SOS Training will also introduce a new set of spells called Field Charms. These can actually be used directly on the map and will have various effects depending on which one you use. For instance, the Trace Charm will reveal more traces nearby whilst the Inn Charm will generate additional spell energy. It has to be cast on Inns, however, as the name would suggest.

There will also be Potion Brewing Enhancements that will boost the strength of your various concoctions alongside reducing the time it takes to make them in the first place. There is also Reward Upgrades which will net players more - and rarer - rewards from Inns, Greenhouses, Fortresses and Family Chests.

To unlock these skills players will have to gather three new resources, Field Guides, Ministry Manuals and Defense Against the Dark Arts Books. The first two will be found by returning Foundables and placing images in the Registry whilst DATDA books will be earned through in-game events.

Harry Potter: Wizards Unite is available now over on the App Store and Google Play. It's a free-to-play game with in-app purchases.
In need of some quality multiplayer games to try with friends? Here are 25 of the very best for iPhone & iPad.
3、A LOVER'S COMPLAINT
4、Love's arms are peace, 'gainst rule, 'gainst sense, 'gainst shame.A DOE blind in one eye was accustomed to graze as near to the edge of the cliff as she possibly could, in the hope of securing her greater safety. She turned her sound eye towards the land that she might get the earliest tidings of the approach of hunter or hound, and her injured eye towards the sea, from whence she entertained no anticipation of danger. Some boatmen sailing by saw her, and taking a successful aim, mortally wounded her. Yielding up her last breath, she gasped forth this lament: "O wretched creature that I am! to take such precaution against the land, and after all to find this seashore, to which I had come for safety, so much more perilous."
- Super Retro World : Pixel Art Maker
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- Temple run 2 MOD APK includes a variety of challenges and individual achievements such as store, objectives, artifacts, leaderboards, coin skins, stats. Essentially the keys will have significant track keys in them before we run into any path. We need to put those keys together. Three keys we need to gather together and open a gold box. We were given one or two of the rewards inside that gold box, and the gift items we needed were cash and emeralds. We can take this game to the next level if we use it to our advantage. Not only will rewards be available, but if we fill in the answers to the questions in that key, the coins will unexpectedly be the only weapons. If we do not want a square-shaped gold coin, we can set up any hole coins in the fish and Old Stone Age. Artifacts This includes more than 50 rewards. Overall we have paths in 5 sections. There are many different types of rewards on all five routes in those five categories.
- SWAT Shooter - Counter-Terrorist strike Mod SWAT Shooter - Counter-Terrorist strike v1.6 mod Features:: Invincible, bullets are not reduced, throwing hands without CD waiting to explode.SWAT Shooter - Retaliatory strike operation.Counter Terrorist Shooter is an epic TPS shooting game. In this war, the terrorists are hiding in a destroyed factory. A brave commando has taken on a difficult mission to go there to destroy all the terrorists. This mission is very challenging, on the battlefield in the face of fierce enemies to defend yourself and destroy the enemy.This is based on a modern shooting game . The goal of the game mission is to fight the terrorists, destroy every enemy and survive in this mission.To attack in silence and the most deadly way to combat the terrorist attacks against terrorists.As an elite commando member, the government has provided you with modern weapons and equipment. Terrorists are planning terrorist attacks. You are assigned to a special counter-terrorist mission. With your excellent fighting skills, kill the terrorists.This action shooter game has many interesting features. These features make this action shooter game totally unique, fun and addictive.In this war, your duty is to fight, shoot, kill, complete the mission. In this war game, prove your shooting skills, fight the enemy. Accept the mission and now download this addictive action shooter game.Features Of Counter-Terrorist Shooting:- HD 3D graphics- Realistic 3D shooting of the battle environment- Easy and smooth operation- a large number of advanced powerful weapons- Radar vision positioning the enemy's position- Best quality attractive sound effects- Play anytime, anywhere
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domain: game


text:
Biden Plans To Suspend New Oil Drilling Permits On Federal Land
Biden's plan will have a massive impact on states such as New Mexico
As part of Joe Biden's plans to crack down on fossil fuels, he is now preparing to implement a moratorium on new oil and gas leasing on federal land.
The plans include a suspension of new drilling permits to oil companies on federal land indefinitely while the Department of the Interior conducts a review of oil development’s impact on climate change.
Biden's plan will have a massive impact on states such as New Mexico, where the federal government controls a third of the state.
The new moratorium is being viewed by Biden fulfilling his promise to end federal land and offshore oil drilling.
But critics have highlighted Biden's repeated promised to “not ban fracking" during his campaign.
Interim Interior Secretary Scott de la Vega froze new oil and gas drilling permits on federal land for 60 days.
The order causes a massive backlash from New Mexico officials and leaders of the Ute Indian Tribe in Utah,
“During his inauguration, President Biden spoke about bringing our nation together. Eliminating drilling on public lands will cost thousands of New Mexicans their jobs and destroy what’s left of our state’s economy,” Dale Janway, the mayor of Carlsbad, New Mexico, said.
“How does that bring us together? Environmental efforts should be fair and well-researched, not knee-jerk mandates that just hurt an already impoverished state.”
Oil industry groups slammed the move.
"This is just the start. It will get worse,'' said Brook Simmons, president of the Petroleum Alliance of Oklahoma.
"Meanwhile, the laws of physics, chemistry and supply and demand remain in effect. Oil and natural gas prices are going up, and so will home heating bills, consumer prices and fuel costs.''
Kierán Suckling, executive director at the Center for Biological Diversity, praised the move.
“The fossil fuel industry has inflicted tremendous damage on the planet. The administration’s review, if done correctly, will show that filthy fracking and drilling must end for good, everywhere,."
Chairman of the Ute Indian Tribe Business Committee, Luke Duncan, wrote in his letter to de la Vega:
The Ute Indian Tribe of the Uintah and Ouray Reservation respectfully requests that you immediately amend Order No. 3395 to provide an exception for energy permits and approvals on Indian lands.
The Ute Indian Tribe and other energy producing tribes rely on energy development to fund our governments and provide services to our members.
Your order is a direct attack on our economy, sovereignty, and our right to self-determination. Indian lands are not federal public lands. Any action on our lands and interests can only be taken after effective tribal consultation.Order No. 3395 violates the United States treaty and trust responsibilities to the Ute Indian Tribe and violates important principles of tribal sovereignty and self-determination.
Your order was also issued in violation (of) our government-to-government relationship. Executive Order No. 13175 on Consultation and Coordination with Indian Tribal Governments, and Interior’s own Policy on Consultation with Tribal Governments.The order must be withdrawn or amended to comply with Federal law and policies. Thank you for your prompt attention to this matter. We look forward from hearing from you.
The Ute Indian Tribe does not mince words in their response to Interior’s order restricting federal energy development:— Megan Barnett Bloomgren (@MeganBloomgren) January 22, 2021
“Your order is a direct attack on our economy, sovereignty, and our right to self-determination.” pic.twitter.com/U5MiZCMx4n


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I have had my F150 a little over a year now. My sync has been acting up since the first week of purchase. I brought it in for repairs the first time and my dealer kept my vehicle for a few days and gave me a loaner car. the second time they blamed it on the navigation system that they had installed, once again they kept the truck I was still paying a note on for a few days pulled the navigation and placed it back in. a day after getting my vehicle back screen started blacking out again.
I found a work around. I would stop car, turn it off, open and closed door and wait a few seconds. This was better than paying a note on a vehicle that I couldn't drive. I did this for several months till it stopped working. I contacted ford and they blamed the dealer for the aftermarket navigation that they installed.
I took my truck back to the dealership. they pulled the navigation out and tested it. they told me to pick my truck up several days later without the navigation. The screen still blacks out and I do not have the navigation system which I paid for. I contacted my lawyer at the time and he said that there wasn't anything that he could do. so I have just been pissed until finding out about this site
- Courtney P.,
Bossier City, LA, US
click to see larger images
The original Backup Camera failed and so the Ford dealer installed a new camera.
As you can see by the attached photo, the second camera is failing as well! There is no excuse for this continued failure. This is not just a fancy option! This is an essential part of driving this vehicle!!
In addition, the entire center console screen (small version) is beginning to fail as well. My wife noticed that when I opened and closed the tailgate the screen turned back on. I will be calling the dealer this week to see what the issue is.
Bloomington, IN, USA


National Report Finds Disabled Left Behind in Emergency and Disaster Plans
by Steven Ertelt
August 12, 2009
Washington, DC (LifeNews.com) — A new report indicates the disabled are left behind when it comes to national emergency and disaster plans, even though President George W. Bush called for better planning in the aftermath of Hurricane Katrina. The Wednesday report from the National Council on Disability is sparking national outrage.
Four patients were alleged to have been killed via euthanasia in the wake of the storm, but two nurses both saw their charges dismissed when they agreed to testify before a grand jury against a physician.
Ultimately, the office of former Attorney General Charles Foti confirmed no charges would be filed against Dr. Anna Pou either when a grand jury refused to return any indictments against her.
The cases prompted more attention to the plight of the disabled in national disasters and emergencies.
It resulted in President Bush issuing an executive order urging federal and local governments, as well as private organizations, to do more to consider the disabled when making such preparations and plans.
But the new report, according to the Washington Times, finds most plans have no provisions for the disabled.
The 500-page report said disaster planners still fail to obtain input from disability groups and cited problems including a lack of service dogs, relocation in trailers and mobile homes, the effectiveness of various warning systems and different transportation needs.
The report is titled "Effective Emergency Management: Making Improvements for Communities and People With Disabilities," and says a lack of disaster help for the disabled is a historic problem. It said plans for help for the disabled "have typically been limited to a few lines in an emergency plan, if they are mentioned at all."
"Although some improvement in this area is evident, catastrophic events such as Hurricane Katrina and the California wildfires exposed the gaps that still exist in many emergency plans and preparedness efforts," the report said, according to the Times. "These events reinforce the need for additional action to protect the lives of people with disabilities against the destructive nature of disasters."
"’Disabilities’ were generally placed into one large category, without consideration for the unique needs associated with each type of disability. Emergency planners often decided what people with disabilities needed without consulting those people," the report added.
Jill Stanek, a pro-life nurse and blogger noted the coincidence of the timing of the report.
"In the midst of a national debate on healthcare rationing of the elderly, disabled, and informed comes this," she said.
She said the news highlighted the differences between Bush and his successor, President Barack Obama.
"The healthcare and emergency care issues are related in spotlighting a constituency Obamacare has been accused of targeting: the disabled," Stanek continued. "Conversely, how many of us would expect Obama to take it upon himself as President Bush did to issue an executive order protecting the disabled in the event of an emergency?"
Going back to Hurricane Katrina, National Public Radio, in February 2006, published papers from the investigation that indicated staff members had a discussion about long-term care patients on the seventh floor and what to do about them.
Three staff members told the attorney general that the plan was to leave no living patients behind and "a lethal dose would be administered" for patients deemed unable to be saved.
One doctor, Bryant King, later told CNN he overheard other conversations.
King refused to identify the people involved in the discussions and later said he never heard them talk about euthanasia, only "ending suffering." Yet the tenor of their discussion led him to believe they were planning to end the lives of patients they deemed beyond hope.
"It appeared they were proceeding with that plan," said Dr. King.
But, Dr. Pou had told Baton Rouge television station WBRZ that "There were some patients there who were critically ill who, regardless of the storm, had the orders of do not resuscitate. In other words, if they died, to allow them to die naturally, and to not use heroic methods to resuscitate them."
"We all did everything in our power to give the best treatment that we could to the patients in the hospital to make them comfortable," Pou said then.
Meanwhile, Angela McManus told AP that her 70 year-old mother was in the hospital at the time recovering from a blood infection and appeared in fine condition when relatives were told to leave the hospital. She died later that day.
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text:
Cosabella Never Say Never Foxie Chemise Black
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Commando Two Faced Tech Half Slip Beige
Commando's revolutionary two-faced tech half slip was designed to keep you flawless. This control women's slip has microfiber on the outside and soft cotton on the inside. Its light-to-moderate compression will leave you smoothed, not stuffed. Fit-tested by real women European microfiber & cotton lining (44% nylon, 43% cotton, 13% spandex...

domain: design


13 April 2018
Jayant’s obsession with creating music, started at an early age. While his friends studied, and his parents worried, he immersed himself in sounds, creating his own unique brand of electronica. His curiosity and eagerness has not waned ever since. He began producing music in 2003 and has done numerous projects since working with artists such as Anoushka Shankar, Chebi Sabah, Karsh Kale, Midival Punditz, to name a few.
Delhi based deep house, techno DJ & producer


text:
Are you a fan of Debbie Macomber and her Blossom Street series?
Her newest ,”Summer on Blossom Street” contains a quote from Joan on page 187!
Email us with the first three words of the quote, your name and email address before August 1st. Your entry will go into a drawing to win a White Lies Designs pattern and a copy of one of Annie Modesitt’s brilliant little Flip technique books! http://www.whiteliesdesigns.com


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I love the Fall season and all the fun decor that comes along with it. I especially love when I can make new and inexpensive things to add to my Fall decorating. I found this cute little jar at Dollar Tree and I thought it would make a great pumpkin!
Disclosure: Some of the links on my site are affiliate links, meaning, at no additional cost to you, I will earn a small commission if you click through and make a purchase. As an Amazon Associate I earn from qualifying purchases.
To make your own pumpkin, you’ll need chalk paint (I found mine at Michael’s) in orange and green along with some antique wax.
Apply 2-3 coats of orange paint to the jar and 2-3 coats of green paint to the lid of the jar, being sure to let the paint dry thoroughly between coats. I like to apply the paint with a chip brush. Once the last coat is completely dry, apply the wax with a chip brush and immediately wipe off the excess with a paper towel. I finished my pumpkin by spraying it with matte sealing spray.
I had raffia in the supply picture above, but decided I liked tying the stem of my pumpkin with natural jute twine better. But of course you can use raffia, ribbon, or even baker’s twine.
And here’s my finished pumpkin! Such a fun addition to my Fall decor. 🍁
Have fun making your own pumpkin – or a whole pumpkin patch! 😉
That’s really cute. I think I might even have a jar like that around here somewhere.
Thank you! Such a fun little project.
Thank you! 🧡
LikeLiked by 1 person

domain: artistic


Updated on July 12, 2018
What is the Definition of Computer Software?
The definition of “software” is not a recently conceived of expression. In the 1935 article, ‘Computable Numbers with an Application to the Entscheidungs Problem, ‘ Alan Turing first proposed and provided the foundation for most modern software. In 1958, the first use of the word software was employed by John W. Tukey in an article in North american Mathematical Monthly. cs gratis
Today, the term ‘computer software’ means written programs, rules, or codes, along with related documentation about the procedure of a computer system that is stored in read/write memory. The “soft” part of software identifies the many products and technology developed using different methods such as programming dialects and scripting languages. The term ‘software’ is much wider and can include different kinds of devices including the procedure systems of modern consumer devices such as vehicles, televisions, toasters, etc.
Computer system software is the job descriptions of exactly what a computer can perform. It can not be physically touched. For illustration, most computers come with software already installed. Computer software is also available on CD where the consumer inserts the disk into the hard drive and follows the installation instruction. Mainly because well, downloadable software is on internet sites such as Microsoft. Computer software is basically a series of instructions that says a computer how to do a particular job. For instance, Microsoft Term is a good example of computer software. As well, web webpage technologies such as CODE, PHP, ASP. NET, and XML are examples of computer software.
Computer programs can be divided into two main categories: operating system software and application software.
Operating-system (0/S): This system involves the fundamental functions that computers perform. An example would be showing motion on a screen for example a mouse cursor was the user moves the mouse button. Types of operating systems include: windows vista, windows vista, Mac, Linux and Unix.
Application Software: These are generally applications that allow the computer to perform the job. Application software can be used to accomplish precise tasks other than just running the pc system. This can include voice, play or record music, view videos, play video games–etc. Various other examples of application software include permitting internet access and printing documents. Application software is the software that indirectly interacts with the computer.
System Applications are the software that can immediately interact with computer hardware. Examples include motherboard, COMPUTER, keyboard, Mouse, printer–etc. Program software is in charge of managing, integrating, and managing the hardware components of a computer system.
Software applications is constructed of machine language which is essentially groups of binary code giving processor chip instructions that are specific to an individual cpu. It is normally written in high-level programming dialects and then interpreted into machine language code. Computer software can be written in assemblage language which is a mnemonic representation of a machine language by using a natural language alphabet.


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Chicago-Area High School Remembers Slain Diplomat
Former teachers of an American diplomat killed in a bombing in Afghanistan have remembered the woman as focused and determined to unravel big problems.
Several of Anne Smedinghoff's former teachers spoke Tuesday at a memorial at the high school she graduated from in the Chicago suburb of Oak Park.
Smedinghoff was one of five Americans killed Saturday in a suicide car bombing while they were delivering textbooks to school children. The Taliban claimed responsibility.
Fenwick High School Spanish teacher Irene Drago says the 25-year-old woman had a gift for foreign languages and a quiet intelligence.
Drago says teachers are supposed to inspire their students, but sometimes students like Smedinghoff come along and inspire teachers.
Smedinghoff continued to visit the school, most recently a few months ago.

domain: news


Kyle Busch continued his march through NASCAR’s record books with yet another victory at Bristol Motor Speedway.
His dominating win Saturday was the 46th of his career in the second-tier Nationwide Series, which puts him three back from tying Mark Martin’s record. But in leading 266 of the 300 laps, he became the first driver in series history to lead more than 10,000 laps.
Busch has led a total of 10,035 laps in the Nationwide Series.
And at Bristol? His win was his 10th spanning all three of NASCAR’s national series, and he’s led a whopping 2,349 laps on the Tennessee bullring.
“Nobody gets around this place better than he does,” crew chief Jason Ratcliffe said.
Busch has won the last four races at Bristol, dating back to August when he had a three-race weekend sweep. He’s got four career Bristol wins in the elite Sprint Cup Series, three in Nationwide and three in the Truck Series.
The Nationwide win moved him closer in his pursuit of Martin’s all-time mark. Martin grabbed a win two weeks ago in Las Vegas, but conceded that Busch would break the mark by this summer. Busch said Martin probably texted him immediately after the race in what’s become good-natured ribbing between the two.
“Mark and I have a little fun egging it on,” he said. “He told me after Las Vegas time is on my side, not his side.”
Kasey Kahne finished second and was followed by Dale Earnhardt Jr., Elliott Sadler and Joey Logano. Kevin Harvick was sixth, Carl Edwards seventh and Jason Leffler, Brad Keselowski and Aric Almirola rounded out the top 10.
Carl Edwards had every reason to believe he had one of the best cars at Bristol Motor Speedway.
Then Goodyear called for a rare tire change in the middle of a race weekend, sending every team back to square one in their preparations for today’s race.
“The tire is a lot slower and it’s going to be a little more difficult to drive,” said Edwards, who is on the pole. “It’s a challenge for everybody.”
Goodyear learned there was a problem when the tires did not lay enough rubber on the track surface. The right-side tires were subjected to considerable wear and lasted only about 30 laps before they began to disintegrate into a powdery substance.
So Goodyear called for nearly 1,300 right-side tires to be shipped to Bristol from North Carolina, and they arrived in time for Saturday’s practice. Teams were given only one set to use over the two practice sessions.
The tire now being used was raced at California and Kansas last year, and has the same outer tread as the tire raced at Bristol last August. But there was no testing data on the tires for use at Bristol, and teams need that to properly set the handling of the car.


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Deviant since Aug 16, 2011 | Premium Member until Mar 25, 2016
This is the place where you can personalize your profile!
By moving, adding and personalizing widgets.
You can drag and drop to rearrange.
You can edit widgets to customize them.
The bottom has widgets you can add!
Some widgets you can only access when you get a premium membership.
Some widgets have options that are only available when you get a premium membership.
We've split the page into zones!
Certain widgets can only be added to certain zones.
"Why," you ask? Because we want profile pages to have freedom of customization, but also to have some consistency. This way, when anyone visits a deviant, they know they can always find the art in the top left, and personal info in the top right.
Don't forget, restraints can bring out the creativity in you!
Now go forth and astound us all with your devious profiles!
Can I use your characters and other work for Role Play sites/blogs for Roleplaying ? NO. Never. Because many of them include characters of other people (commissions, trades etc), which I don't have ownership of. And I don't want my characters to be used by other people either.
Can I highly reference/trace from your work / copy parts of your characters? NO. Especially not copying my characters! I don't want to be whiny. But please make your own. It took me 8 years to refine most of my characters their designs and I don't like seeing 8 years of work being -TAKEN- by others. If you want to use my work to PRACTISE drawing, then I do allow it as long as you credit me. But again, no direct copying of characters or complete works.
Can I post your fan art of existing anime/books/tv series on my blog/site/etc.? Yes, but only if you link back to me deviantart account and/or the drawing in my gallery. This is the ONLY exception I make when it comes to posting my work on another site.But you are NOT allowed to sell the work, trace it, highly reference it etc. So if you are keeping a fan blog of let's say FMA or Bleach. Then you are allowed to post the drawing WITH a LINK to my account/the original drawing.
Can I share your work on Pinterest / Tumblr ? Yes, as long as you link back to my deviantART page or the original drawing in a way.
Can we be friends? Well, Becoming friends isn't done by simply asking. Maybe it worked when we were little kids and simply asked the other "shall we be friends"? The other would say yes. But, we're grown up people and friendship is something that grows. It's something build on trust, having fun, sharing eachothers good and bad times and so on. I won't ramble on about what friendship is. But don't expect me to say yes if you expect us to be friends by simply asking xD It's something you have to work on. BTW. Becoming a friend of mine is NOT going to get you free art from me, if that's your reason to get close to me, get the fock off my page (: The same for drawing me gifts, yes I appreciate it but it's not going to guarantee you getting anything drawn as well from me. Gifts are voluntary, some people don't understand that.
Do you do requests? / Can you draw this for me? No, It would be unfair towards the people who commission me.
Can you teach me to draw like you / give me private lessons in art? First of all.. I'm not a teacher :,D And I don't have the time to give every single person who asks me private lessons. Plus, there's no way you're ever going to draw the same way as another person for those who want to draw like a certain artist. It's more important to discover the world of art by yourself with TIPS of other artists... but know that they won't have time to give you personal lessons. You could always follow a drawing class near the place you live though who are given by people who's job it is to teach about art and drawing.
Can you look at my gallery / art ? Maybe... but realize dozens of people ask me this on a regular basis and also ask for critique. It depends on if I have enough time. But realize I'm a pretty busy person :/ If you want comments/critique from me. I advise you think about what EXACTLY you want my critique on and if you are looking for help with drawing, I advise you sent me a seperate note with the exact drawing with an exact description of what you want me to help with. BUT DON'T RANDOMLY ASK ME TO LOOK. It's annoying :,|
Will you please watch me / give me points / give me other things? Again... don't ask me for stuff >.< I don't give points unless it's for a VERY GOOD reason. If one of your pets or family members are in danger well then I will. But not if it's because you simply want more points or other stuff :/ And I don't respond to questions like "wil you please watch me". Know that it doesn't work that way -,- If I like your work and if it inspires me, then I will.
Can I draw one of your characters? I consider these drawings gifts, so yes of course ! :,D But still, as long as you credit me for the character and not claim them as your own.
Can we RP / Do you RP? Nope. I am simply not into it and I also don't have the time to do so.
What drawing programs do you use? Photoshop CS6 and Paint Tool Sai.
What tablet do you use to draw digitally? I use a Wacom Intuos3 Oversize. And as for the fillers I use for my pen, I use felt-tip fillers (so not the regular ones).
What do you use to sketch? It varies. I use my Bic Cristal ballpoint pens the most though when it comes to sketching traditionally. I still use the Pentel mechanical pencils a lot as well. And recently I started sketching with the Pentel Pocket Brush as well. And sometimes, I also sketch in Paint Tool Sai or Photoshop CS6.
What canvas size do you use for digital work? I work on 5000px or above for most big digital drawings. Some sketches or small drawings are often done on smaller canvas sizes though.
What inspires you to draw what you draw ? I get inspiration from pretty much anything and anywhere. I mostly get inspired by various cultures (mostly eastern) and nature (landscapes, clouds, animals etc), by people I've seen in my life or who are close to me, various anime series and artists I look up to have also inspired me for a huge part, and of course a lot of artists here on deviantArt who inspired me to start drawing (;
Do you go to art school ? Nope, I once applied to go to one but I changed thoughts and went in another direction to study Urban Design (: I've always been and still am a self-taught artist.
How long have you been around on deviantART? On my old account 5 years or so, here 3.. so around 8 years total.
How long have you been drawing? I was drawing before I was writing : P But in a serious way... for about 10-11 years or so.
Do you have skype / can we chat ? Yes I do have skype, THOUGH I'm hardly online for chitchat / I don't use it much as I pretty much always have got something to finish or do so I practically don't have time to chat online :,D I use it more to discuss trades and collaborations / as a form of work-communication.
Why don't you do livestreams? My workflow is very unstable. I take breaks A LOT. And on workdays I often only have around 15-30 minutes to work on a drawing. My way of working just doesn't fit the whole livestream thing :/ I do make videos though and post them on my Youtube account.
Are you planning on making a comic? Nope, sadly I don't have the time to do so in my life. Real life stuff is keeping me way too busy to actually set up a full comic. I do write plots for my stories though and draw out various scenes/moments and characters to visualize their world. But still, not a full comic.
Why don't you reply to my comment / note ? I'm probably stuck in a busy period in real life. And due to stuff going on with college and work in real life, it might take me longer (couple of days) to answer. I will most likely answer in weekends.
Music = Life
S e n p a i
N A V I G A T I O N
A R T W O R K - U S A G E
S t a t u s
S T A T U S
K I R I B A N 213.456
T R A D E S: CLOSED P.S. I do not make promises. But I do make exceptions if I really happen to like your work (;
C O M M I S S I O N S: CLOSED
C O L L A B O R A T I O N S: CLOSED Only for people with the same level of skill (and preferably a similar way of drawing/style/etc). It's not to discriminate... it's to let the collaboration look balanced.
R E Q U E S T S: CLOSED I don't do them, it would not be fair towards my commissioners.
T O - D O Collaboration with dNiseb Collaboration with DavesPineapple PDF files with biographies of my characters PDF file with a elaborate description of Daniel's world
R E A L - L I F E - A G E N D A 9th February - 1st July : On internship, working 40 hours a week + writing report in free time for school. Won't be active much in those 5 months with art/will be slow with replying sometimes. 8th June - 16th June : Week of having to deliver my report of my internship + will be stressing to work on a presentation for my graduation. Will be inactive and reply very slow to messages or even only after the 16th of June. 21th July - 24th July: Will be on a "holiday" in my own country to experience the "March of Nijmegen". I won't be active online those days. 23th of July : My birthday ! I'm celebrating it during the March of Nijmegen so I'll probably be active again after those days. 29th of July: On a holiday abroad for 12 days. I won't have Wi-Fi all the time so I'll probably answer when I'm back from my holiday.


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Countless scientific studies have been conducted in the name of immunology, the study of human immunity. Scientists look to the immune system for a better understanding of asthma, allergies, cancer, metabolic diseases, and many other health conditions. Likewise, researchers are seeking to understand how and why the immune system malfunctions in some individuals. Abnormal activation of the immune system can point to many different diseases that cause chronic inflammation, tissue destruction, and more. Autoimmune disease (AD) and autoinflammatory disease (AIF) are two categories of illnesses that occur when the immune system mistakenly damages healthy cells. Although these inflammatory disorders share many commonalities, there are key features that make them unique.
In order to describe the differences between autoimmune disease and autoinflammatory disease, it’s necessary to understand a few key facts about immunological function. The human immune system is a complex network of components that work to neutralize infections and protect the body from disease. Within the immune system are two lines of defense: innate immunity and adaptive immunity . The innate immune system includes defensive barriers such as the skin and mucous membranes, as well as an army of chemicals and cells that target disease-causing pathogens [2,3].
When provoked, the innate immune system releases an onslaught of these defensive properties. Some innate cells also secrete cytokines, which are proteins that deliver signals between immune cells. Additionally, cytokines play an important role in regulating fever and inflammation during an immune response . Innate immunity can mobilize quickly, usually within minutes to hours of encountering a threat. Activity from an innate immune response summons the body’s second line of protection, known as the adaptive immune system .
When provoked, the innate immune system releases an onslaught of these defensive properties.
Both innate and adaptive immunity can be activated by antigens, which are molecular structures that exist on the surface of human and pathogen cells . However, innate immunity can only identify the presence of foriegn antigens. Adaptive immunity recognizes specific foreign antigens and also builds specialized defenses against them. T cells and B cells are key players in the adaptive immune response . Together, these cells “memorize” the antigens of infectious agents, allowing the immune system to build a swift and targeted defense against repeat infections . Adaptive immune cells are hard-wired to ignore host cell antigens. Known as self-tolerance, this mechanism prevents the immune system from attacking healthy human tissues .
Most distinctly, autoinflammatory diseases only involve the innate immune system. AIFs occur when innate immune cells are activated without an infection or injury being present. This mishap kickstarts the release of cytokines and other immune responses, causing fever and inflammation. Recurring episodes of high fever are the primary symptom of AIF . Similar to some autoimmune diseases, autoinflammatory symptoms can be widespread, affecting the joints, gastrointestinal tract, skin, eyes, and internal organs.
Autoinflammatory diseases are caused by mutations in the genes that control and regulate innate immune cells . Some AIFs, including Familial Mediterranean Fever (FMF) and TNF receptor–associated periodic syndrome (TRAPS), are known as monogenic because they are caused by a singular known genetic defect [12,13]. Because these diseases originate in the genes, they can run in families .
Autoinflammatory syndromes are very rare. Presently, there is no known gender prevalence . Unlike autoimmune disease, the majority of AIFs develop during childhood. Due to the rarity of these diseases and their close appearance to other illnesses, diagnosis is often delayed . Several AIFs are known for their higher frequencies within certain ethnicities. As such, age of onset and familial history, in addition to clinical observations, can be helpful in diagnosing these rare conditions. In some cases, testing for gene mutations is used to confirm a diagnosis of AIF [12,16,].
While autoinflammatory diseases are caused by problems with innate immunity, autoimmune disease originates in the adaptive immune system. AD occurs when adaptive immune cells lose their ability to maintain self-tolerance of human cells. As a result, the immune system begins attacking healthy tissues as though they were infectious agents. Some ADs are organ-specific, whereas others can affect multiple parts of the body. Like autoinflammatory syndromes, recurrent fever and chronic inflammation can be attributed to autoimmune disease. However, many possible symptoms are unique to AD, including hair loss, dry mouth, temperature sensitivity, muscle weakness, reproductive issues, and more.
Researchers believe that a variety of conditions including environment, hormones, injury, infection, and genes could all play a role in initiating autoimmunity.
AD is thought to be triggered by a combination of multiple factors not limited to genetics. Although the exact causes remain unknown, researchers believe that a variety of conditions including environment, hormones, injury, infection, and genes could all play a role in initiating autoimmunity. Numerous investigations demonstrate multifactorial causes of AD, including a Johns Hopkins study on patients who developed Scleroderma at the same time that their immune system was fighting cancer cells . Another example is psoriasis, an autoimmune skin condition that often appears within a short time after a strep throat infection .
In contrast with autoinflammatory disease, autoimmune conditions are quite common. Current data estimates that ADs affect at least 14 million people in the U.S. alone. There are 80-100 known autoimmune and autoimmune-related conditions, including Hashimoto’s thyroiditis, Type 1 diabetes, Sjögren’s, and celiac disease. Gender prevalence in AD is well documented. In fact, around 80% of those diagnosed are women. While ADs can affect anyone at any age, the majority tend to appear during adulthood .
One challenging aspect of diagnosing an AD is that symptoms can vary widely- even amongst individuals with the same disease. Neither AD nor AIF has a standard presentation. This is just one reason why diagnosing these conditions can be a lengthy and complicated process. It also underscores the need for further scientific investigations and efforts to promote awareness.
Although it is helpful to differentiate inflammatory disorders based on their involvement with innate or adaptive immunity, some conditions have combined features of autoimmune and autoinflammatory disease. As such, many researchers note that AIF and AD, rather than being two distinct groups, could actually be part of a spectrum of inflammatory disorders [22, 23]. Diseases that involve both types of immunity include ankylosing spondylitis (AS), psoriatic arthritis, Behcet’s disease, Crohn’s disease, and more .
Autoinflammatory and autoimmune conditions belong to a complex group of diseases characterized by immune system dysregulation. While scientists continue to search for answers involving the mechanisms of these diseases, it is clear that immune-related illnesses are becoming more prevalent in Western nations. Adding to the enormous importance of studying these conditions, chronic inflammation is linked to numerous other health concerns, including heart disease, cancer, stroke, and more . In order to improve strategies for diagnosis, treatment, and patient support, it is key that researchers continue their commitment to improving knowledge about inflammatory disorders.
Warrington, R., Watson, W., Kim, H.L. et al. (2011). An introduction to immunology and immunopathology. All Asth Clin Immun.
Institute for Quality and Efficiency in Health Care. (2006). The innate and adaptive immune systems.
Molnar, C., & Gair, J. (2015). Concepts of Biology – 1st Canadian Edition. BCcampus.
Arango Duque, G. & Descoteaux, A. (2014) Macrophage cytokines: involvement in immunity and infectious diseases. Front. Immunol.
Cano, R.L.E. & Lopera, H.D.E. (2013). Introduction to T and B lymphocytes. El Rosario University Press.
Thompson, A.E. The Immune System. JAMA.
Chaplin, D.D. Overview of the immune response. (2010). J Allergy Clin Immunol.
National Institute of Allergy and Infectious Disease. (2014). Immune Tolerance.
The Walter and Eliza Hall Institute of Medical Research. (2021). Autoinflammatory Diseases.
British Society for Immunology. Autoinflammation.
Cush, J.J. (2013). Autoinflammatory syndromes. Dermatol Clin.
Masters S.L., Simon A., Aksentijevich I., Kastner D.L. Horror autoinflammaticus: the molecular pathophysiology of autoinflammatory disease. (2009). Annu Rev Immunol.
De Sanctis, S., Nozzi, M., Del Torto, M. et al. (2010). Autoinflammatory syndromes: diagnosis and management. Ital J Pediatr.
Navallas, M., Inarejos Clemente, E.J., Iglesias, E. et al. (2020). Autoinflammatory diseases in childhood, part 1: monogenic syndromes. Pediatr Radiol.
Mulders-Manders, C.M., van der Hilst, J.C.H., van der Meer, J.W.M., & Simon, A. (2019). Systemic Autoinflammatory Syndromes. Clinical Immunology (Fifth Edition).
McGonagle D., McDermott M.F. (2006) A Proposed Classification of the Immunological Diseases. PLoS Med.
Caso F., Galozzi P., Costa L., et al. Autoinflammatory granulomatous diseases: from Blau syndrome and early-onset sarcoidosis to NOD2-mediated disease and Crohn’s disease. RMD Open.
Darrah, E. (2017). Systematic autoantigen analysis identifies a distinct subtype of scleroderma with coincident cancer. Johns Hopkins Rheumatology.
Autoimmune Disease: Why Is My Immune System Attacking Itself?. Johns Hopkins University.
Angum F, Khan T, Kaler J, Siddiqui L, Hussain A. (2020). The Prevalence of Autoimmune Disorders in Women: A Narrative Review. Cureus.
El-Shebiny, E.M., Zahran, E.S., Shoeib, S.A. et al. (2021). Bridging autoinflammatory and autoimmune diseases. Egypt J Intern Med.
Rosenbaum, J. Spondyloarthritis: Autoimmune or Autoinflammatory? Spondylitis Association of America.
Mauro, D., Thomas, R., Guggino, G. et al. (2021). Ankylosing spondylitis: an autoimmune or autoinflammatory disease?. Nat Rev Rheumatol.
Arakelyan A, Nersisyan L, Poghosyan D, et al. (2017). Autoimmunity and autoinflammation: A systems view on signaling pathway dysregulation profiles. PLoS One.


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Targim is a large medieval tower castle settlement. It lies on the right bak of river Assa. There are 4 battle and more than two dozens of dual-purpose and residential towers with stone outbuildings here, all at different stages of decay. The battle towers rise for 30 meters into the sky.
In the past, the village was famous for its talented battle tower builders, experienced gunsmiths and dashing mounted warriors. According to a popular legend, in ancient times there lived in Egikal a particularly respected man named Alberd. He had a large family: three sons – Egi, Hamkhi, Targim, - and four daughters – Aiset, Baiset, Mislimat and Mislinat. Alberd was related to a noble Georgian family. Before his death, Alberd made a will, according to which the eldest son, Egi, got the father`s house, Hamkhi had to live in Khama and Targim on the right bank of Assa. After his father`s death, Hamkhi got married and settled on the place where now the village of Hamkji lies. Here, two versts south of Egikal, he founded a new settlement that was named after him. Hamkhi`s brothers and sisters helped him to build the place. Together, they built a residential tower and the necessary outbuildings. Then they came together to a home for Targim, the youngest and favourite son of Alberd. On the right bank of Assa, where the father prescribed, brothers and all their relatives built battle and residential towers. Having finished construction operations, Targim with his mother and sisters leaves the father`s house and moves to a new location, which later became known as the village of Targim.
Tourist Information Centre of the Republic of Ingushetia /www.ingtourism.ru
|Address:||Republic of Ingushetia, Dzheirakh district|

domain: geography


Which means that, a minimum of at first, the Buick Grand National 2020 will probably be bought with a 2.3-liter four-cylinder engine. The itemizing doesn’t specifically verify that it’s an EcoBoost mill, however that is very likely given that the Bronco will have the identical underpinnings because the Ranger truck.
A fast look at Ford’s current automobile lineup is enough to notice that the brand’s mannequin do a number of sharing. Which means that the Buick Grand National 2020 will function the exact same engine as the Ranger. This specific 2.3-liter EcoBoost mill cranks out 270 horsepower and 310 pound-feet of torque. That’s removed from large, however it’s not bad either given the scale of the SUV. And that torque determine is definitely impressive.
The mill contains a twin-scroll turbocharger that extracts exhaust gasoline from two pairs of cylinders in alternating sequence so as to improve pulse power and scale back lag. It additionally options an Auto Start-Stop perform that shuts off the engine when the automobile comes to a stop and restarts it immediately when the brake is released. This feature improves gasoline economy. The Ranger is rated at 21 mpg city, 26 mpg highway, and 26 mpg combined, figures that shouldn’t change a lot for the Bronco.
Buick Grand National 2020 pairs the two.3-liter EcoBoost to a 10-speed automated transmission in the Ranger, and this unit should carry over to the Bronco. A brand new design, the SelectShift gearbox features optimized gear progressions for smooth shifting.
Buick Grand National 2020 is about to take the pickup world by storm with an all-electric truck that will probably be revealed this yr. The automobile that’s set to be shown this yr gained’t be the final production-spec model, though, as we’ve come to expect from Buick Grand National 2020, but it will be a really close preview of that model.
Buick Grand National 2020 All fashions of the Tesla Pickup will likely function a dual-motor setup and therefore be all-wheel drive. Since the battery pack will likely be built-in into the floor of the truck, it should have a really low middle of gravity for a car of its sort. So, it would in all probability be fairly steady and nice for off-road hooning where it should feel stable and not prone to tipping over (like some conventional vans are inclined to feel when pushed to make onerous turns).
Buick Grand National 2020 It might very nicely be that Tesla is designing this pickup so that it could possibly taxi heavy spacecraft around runways, to and from the launch pad. We’re certain there’s a catch to that ridiculous 300,000-pound declare, but either wither way, the pickup may have a higher towing rating than all other conventional pickups – perhaps this is what Elon was attempting to recommend.
Attempting to make a tall, heavy pickup truck handle like probably the most iconic sports activities automobiles on the planet appears right on unattainable. Maybe between its huge energy and torque figures and the aforementioned energetic suspension whose job might not only be to adjust the ride height based on load, but in addition help the truck nook flat, it will likely be the most effective handling truck ever made; beating a Porsche around corners seems like a far much less likely state of affairs, although.
Buick Grand National 2020 Relating to the look of the Tesla pickup, we now have very little to go on by way of speculating what it’d appear like. The automaker just lately launched a teaser exhibiting a light bar that runs the width of the automobile, however we don’t know if it exhibits the front or the rear of the car (just like the Rivian R1T pickup). If it’s the rear, then the bed has a lid that fully closes it off and turns it into a trunk. If it’s the front, then the truck may have a single LED strip catering for all front end illumination wants Buick Grand National 2020.
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Of course, you’ll find overall and field-related Cal state long beach geology. Common books such as how to compose an essay, critical thinking, and how to take care of competitiveness in getting a qualification can help you a good deal during your program. Regarding that field-related books, you then just have to decide on the main one suited for your subject of analysis. If you are a literature studentthen get some good books on prose, drama, or background of literature. If you’re in a doctoral course, then you definitely can read various anatomical books along with the human body books. The previous thing is you need to find a book that is affordable accordingto the available budget.
Another Cal state long beach geology is The Power of Habit: Why We Do What We Do in Life and Business from Charles Duhigg. The book shows us how to adjust our own lives by simply modifying our dependence. It’s an ideal book for faculty pupils to enter the following chapter in your own lifetime. The book teaches usespecially college pupils, the best way to develop a excellent custom and also just how exactly to break a bad behavior. Every productive men and women have a excellent practice. Hence, you have to form a excellent habit if you want to be as powerful as them.
Cal state long beach geology for Q Training
Subsequently, the Cal state long beach geology have traces and checkered patterns. Handwriting books who’ve checkered lines are ideal for all those who are analyzing mathematics or engineering. The lines may help it become easier that you write amounts and draw on graphics. In addition, these among you that are studying Japanese or Chinese, such a notebook is also fantastic for educating Japanese and Chinese vocabulary in order that the composing looks neater.
The next of best Cal state long beach geology which you have to read is your Old Man and the ocean. This story is loosely based on mcdougal encounter when he was in Cuba. The narrative , he generated an older fisherman who sailed alone in his tiny boat but stopped captured up a sizable Marlin. Alas the marlin has to be destroyed by the parasite that is senile.