MiR-145 functions as a tumor suppressor in Papillary Thyroid Cancer by inhibiting RAB5C.

Papillary thyroid carcinoma (PTC) accounts for the largest proportion of thyroid cancers; and its morbidity rate has dramatically increased in recent decades. However, the pathogenesis mechanisms of PTC are still not clear. This study aimed to reveal that miR-145 acts as an antitumor miRNA in the progression of PTC. In the present study, the expression of miR-145 was analyzed in 57 paired PTC patient samples. The relationship between clinicopathological features and miR-145 expression were also defined. The tumor suppressive function of miR-145 on PTC cell metastasis, proliferation and apoptosis were revealed in vitro. Also, we used dual luciferase reporter assay to define the relationship of miR-145 and RAB5C. RAB5C was reported to participate in cell invasion and cell motility. We found that miR-145 was downregulated in PTCs, which was negatively correlated with PTC progression and metastasis. MiR-145 inhibited PTC migration, proliferation and promoted apoptosis by directly suppresing RAB5C. In conclusion, miR-145 functions as a tumor suppressor in PTC by inhibiting RAB5C. MiR-145 and RAB5C are potential therapeutic targets in therapy of aggressive PTC cases.

Key modules and hub genes identified by coexpression network analysis for revealing novel biomarkers for larynx squamous cell carcinoma.

Larynx squamous cell carcinoma (LSCC) is the second most aggressive head and neck squamous cell carcinoma. Numerous genes have been identified to be aberrantly expressed during the development of LSCC. However, currently, researchers focus more on the individual molecule and downstream genes, leaving the coexpression among genes and key upstream disease driver genes unexploited. In this study, we applied weighted gene coexpression analysis (WGCNA) to decipher potential hub genes driving the development of LSCC. After downloading of LSCC microarray profile from gene expression omnibus, different expression analysis was performed, which was used to conduct functional enrichment analysis. Then, we applied WGCNA to highlight the hub genes which were relevant to the carcinogenesis and progression. A total of 2858 differentially expressed genes were identified in LSCC samples compared with adjacent non-neoplastic tissues. WGCNA revealed three LSCC set-specific modules having significant Kyoto Encyclopedia of Genes and Genomes enrichment effect, including pink, cyan, and black module. Nine hub genes were identified to be crucial in LSCC onset and progression, which may assist clinical decisions and serve as potential targets for LSCC treatment.

MiR-155 promotes anaplastic thyroid cancer progression by directly targeting SOCS1.

BACKGROUND: Anaplastic thyroid cancer (ATC) is considered to be a rare type of thyroid cancer but takes up the most important proportion of thyroid cancer-related deaths. Therefore, the development of molecular targeted therapy is an exciting strategy in the management of ATC. METHODS: miR-155 and SOCS1 expression were measured by qRT-PCR as well as western blot analysis. 8305c and FRO cells were transfected and cultured for apoptosis assays, transwell, MTT on miR-155 or SOCS1 suppression and overexpression. Dual-luciferase reporter assays and SOCS1 restoration experimentswas implemented for define the relation between SOCS1 and miR-155. In addition, the correlation between miR-155 expression and patients' clinicopathological features were also explored. RESULTS: Aberrant miR-155 and SOCS1 expression and inverse correlation were found in ATC samples. In addition, it indicated that miR-155 expression correlated with cervical metastasis as well as extrathyroidal invasion. Moreover, we demonstrated that miR-155 inhibited 8305c and FRO cells apoptosis, promoted proliferation, invasion and migration. Furthermore, miR-155 inhibition was associated with a significant overexpression of SOCS1. Additionally, luciferase reporter assays presented that miR-155 could bind to SOCS1 3'-UTR, influencing its stability negatively and finally lowering SOCS1 levels. Moreover, it was illustrated that the impacts of miR-155 suppression were reversed by the inhibition of SOCS1 on cell proliferation, apoptosis as well as invasion. CONCLUSIONS: Aberrant miR-155/SOCS1 expression has been included in ATC progression: miR-155 overexpression leads to SOCS1 suppression and develops ATC progression. Thus, miR-155 has been considered to be an underlying therapeutic target for ATC.

miR-196b is a prognostic factor of human laryngeal squamous cell carcinoma and promotes tumor progression by targeting SOCS2.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) has the second highest incidence among the head and neck malignancies. Additionally, the incidence of LSCCs has been recently increasing. Therefore, understanding the mechanisms of LSCC tumorigenesis and identifying novel biomarkers to accurately predict and improve the prognosis of patients with LSCC is extremely important. METHODS: miR-196b and SOCS2 expression was measured by qRT-PCR and western blot. Their correlation was analyzed with the Pearson test. TU212 and TU177 cells were cultured and transfected for MTT, Transwell, and apoptosis assays upon miR-196b knockdown, SOSC2 overexpression or SOCS2 silencing. Dual-luciferase reporter assay were conducted to identify the relationship between miR-196b and SOCS2. Moreover, the correlation between clinicopathological parameters and miR-196b/SOCS2 expression in patients was analyzed. Univariate and multivariate analysis and log-rank tests were used to determine if miR-196 was an independent LSCC prognostic factors. RESULTS: We reported the aberrant expression and inverse correlation of miR-196b and SOCS2 in LSCC samples. miR-196b promoted LSCC cells proliferation and invasion, and suppressed apoptosis by directly inhibiting SOCS2 expression in vitro. Moreover, we also revealed that miR-196b/SOCS2 expression correlated with T stage and cervical metastasis. miR-196b was demonstrated to be an independent prognostic factor for overall survival of patients with LSCC. CONCLUSIONS: Overexpression of miR-196b suppresses SOCS2 in human LSCC resulting in tumor progression and poor prognosis. miR-196b is a potential marker for prognosis assessment and targeting miR-196b may be a novel valuable strategy for the treatment of LSCC.

YB-1 promotes laryngeal squamous cell carcinoma progression by inducing miR-155 expression via c-Myb.

AIM: In this study, we investigated the role of Y-box binding protein-1 (YB-1), c-Myb and miR-155 in human laryngeal squamous cell carcinoma (LSCC) progression. MATERIALS & METHODS: Quantitative real-time PCR, western blot, MTT and Transwell were conducted to determine the expression and function of YB-1/miR-155 pathway. Univariate and multivariate analyses were used to determine the prognostic factors. RESULTS: Expression of YB-1, c-Myb and miR-155 was higher in LSCC tissues. YB-1 promoted proliferation, invasiveness and migration of Hep-2 cells in vitro. Patients with higher YB-1 correlated with advanced T stage, poor differentiation and cervical metastasis. LSCC patients with high YB-1 expression showed poor overall survival. CONCLUSION: YB-1 promotes LSCC progression by increasing miR-155 levels via c-Myb and acts as a prognostic factor.

miR-181a targets GATA6 to inhibit the progression of human laryngeal squamous cell carcinoma.

AIM: We sought to determine the function of miR-181a/GATA6 pathway in the progression of laryngeal squamous cell carcinoma (LSCC). MATERIALS & METHODS: The expression of miR-181a and GATA6 were detected using quantitative real-time-PCR and western blotting in 127 LSCC samples and 32 corresponding control mucosa tissues. Cell death, migration and apoptosis were measured in Hep-2 cells using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), Transwell migration assay and apoptosis assay, respectively. The prognosis was determined by the follow-up, univariate analysis and multivariate analysis. RESULTS: We observed decreased miR-181a levels and increased GATA6 expression in LSCC samples compared with control mucosa tissues. Transfection of miR-181a decreased GATA6 expression, suppressed migration and promoted apoptosis in Hep-2 cells. Furthermore, silencing GATA6 suppressed cell migration and promoted apoptosis in Hep-2 cells. Notably, patients with high miR-181a levels had a longer life span. CONCLUSION: MiR-181a inhibits LSCC progression via suppressing GATA6 expression. MiR-181a is an independent prognostic factor in LSCC patients.

The role of HOXA9 in human laryngeal squamous cell carcinoma.

The present study was performed to investigate the expression of HOXA9 in human laryngeal squamous cell carcinoma and its possible roles in the progression. The levels of HOXA9 mRNA and protein were evaluated in human laryngeal squamous cell carcinoma. Hep-2 cells were transfected with h-HOXA9-siRNA. CCK-8 was used to analyze cell proliferation. Flow cytometry (FCM) was used to analyze cell cycle. The mobility of cells was tested by transwell migration assay. The expression of HOXA9 in laryngeal squamous cell carcinoma was significantly higher than normal mucosa tissues. In in vitro experiments, downregulation of HOXA9 strongly inhibited cell growth in Hep-2 by arresting cells in G1 phase (p < 0.05). Transwell migration assay showed that more HOXA9-negative cells migrated to the lower side of the membrane than positive ones (p < 0.01). HOXA9 acts as an oncogene in laryngeal squamous cell carcinoma. It could promote the proliferation and migration of Hep-2 cells.

Antitumor effects of Dasatinib on laryngeal squamous cell carcinoma in vivo and in vitro.

A novel drug named Dasatinib is a highly potent ATP-competitive orally active dual Src/Abl kinase inhibitor with anti-proliferative activity against solid tumors and CML (chronic myeloid leukaemia) cell lines. Dasatinib has been shown to have preclinical activity against human prostate, breast, pancreatic, lung, and head and neck cancer. To determine whether Dasatinib can inhibit the growth of laryngeal squamous cell carcinoma, in the present study, we investigated the antitumor effect of Dasatinib on Hep-2 cells. Hep-2 cells were treated with different concentrations of Dasatinib for different time. Cell proliferation, cell cycle distribution, and cell apoptosis were evaluated using MTT assay, flow cytometry, and fluorescent microscopy. It was found that Dasatinib exhibited significant efficacy in growth inhibition, cell cycle arrest at G0/G1 phase, and apoptosis induction in a dose- and time-dependent manner. Measuring the modulation of regulators in the cell cycle, apoptosis and signal transductions by western blot analysis showed that the effect of Dasatinib was due to suppression of the expression of Bax, Bcl-2, Caspase-3, and Caspase-8. Moreover, in vivo studies were performed in a nude mouse xenograft model, the new prescription (DDP + Dasatinib) was better than DDP alone in terms of therapeutic efficacy. In conclusion, the antitumor effect of Dasatinib on Hep-2 cells was due to the induction of cell cycle arrest as well as apoptosis. The possible mechanisms underlying the action might be attributed to the suppression of Src phosphorylation. This investigation suggests a potential clinical application of Dasatinib for the treatment of laryngeal cancer patients.

Antitumor and immunoregulatory effects of astragalus on nasopharyngeal carcinoma in vivo and in vitro.

This study was carried out to evaluate the effects of Astragalus on human nasopharyngeal carcinoma (NPC) viability and apoptosis and to investigate the mechanism of Astragalus in a NPC cell line (CNE2). Cell viability was measured using the MTT assay. CNE2 cells treated with Astragalus were stained with acridine orange/ethidium bromide and subjected to fluorescence microscopy. Bcl-2, Bax, caspase-3 and -8 were measured by western blotting. Rat NPC cells were used to establish a NPC model. Tumor weight, immune organ index and T lymphocyte subsets were employed to detect the immunoregulatory and antitumor effects of Astragalus after administration. Astragalus was effective in inducing apoptosis in CNE2 cells. Morphological changes associated with cell injury were found. Western analysis showed caspase-3, -8, and Bax protein levels were increased after Astragalus treatment, while the bcl-2 protein level was decreased. Astragalus increased the percentage of CD3(+) , CD4(+) T-lymphocytes, and the ratio of CD4(+) /CD8(+) . Astragalus also restored the immunological effects of DDP-induced immunosuppression. These findings suggest that the immunomodulatory and anticancer effects of DDP + Astragalus were better than those of DDP alone, and Astragalus could inhibit immunosuppression induced by DDP. The combination of CDDP + Astragalus could be developed as an effective chemotherapeutic regimen in the treatment of nasopharyngeal carcinoma.

Development and Validation of an Immune-Related Signature for the Prediction of Recurrence Risk of Patients With Laryngeal Cancer.

OBJECTIVE: Our purpose was to develop and verify an immune-related signature for predicting recurrence risk of patients with laryngeal cancer. METHODS: RNA-seq data of 51 recurrence and 81 non-recurrence laryngeal cancer samples were downloaded from TCGA database, as the training set. Microarray data of 34 recurrence and 75 non-recurrence cancer samples were obtained from GEO dataset, as the validation set. Single factor cox regression was utilized to screen prognosis-related immune genes. After LASSO regression analysis, an immune-related signature was constructed. Recurrence free survival (RFS) between high- and low- recurrence risk patients was presented, followed by ROC. We also evaluated the correlation between immune infiltration and the signature using the CIBERSORT algorithm. The genes in the signature were validated in laryngeal cancer tissues by western blot or RT-qPCR. After RCN1 knockdown, migration and invasion of laryngeal cancer cells were investigated. RESULTS: Totally, 43 prognosis-related immune genes were identified for laryngeal cancer. Among them, eight genes were used for constructing a prognostic signature. High risk group exhibited a higher recurrence risk than low risk group. The AUC for 1-year was separately 0.803 and 0.715 in the training and verification sets, suggesting its well efficacy for predicting the recurrence. Furthermore, this signature was closely related to distinct immune cell infiltration. RCN1, DNAJA2, LASP1 and IBSP were up-regulated in laryngeal cancer. RCN1 knockdown restrained migrated and invasive abilities of laryngeal cancer cells. CONCLUSION: Our findings identify a reliable immune-related signature that can predict the recurrence risk of patients with laryngeal cancer.

Long noncoding RNA LINC01638 contributes to laryngeal squamous cell cancer progression by modulating miR-523-5p/BATF3 axis.

Long noncoding RNA (lncRNA) plays a critical role in tumorigenesis. How lncRNA regulates laryngeal squamous cell carcinoma (LSCC) progression remains poorly understood. In the present study, we found that LINC01638 was highly expressed in LSCC tissues. And LINC01638 expression was positively correlated with clinical stage and lymph node metastasis. Patients with LINC01638 high expression displayed a low survival rate. Results from CCK8, colony formation, and transwell assays showed that LINC01638 knockdown suppressed the proliferation, migration and invasion of LSCC cells in vitro. Animal experiments indicated that LINC01638 silencing attenuated tumor growth in vivo. In terms of mechanism, LINC01638 was found to sponge miR-523-5p and promote BATF3 expression. In summary, our results demonstrated that LINC01638/miR-523-5p/BATF3 axis plays a crucial function in initiating LSCC development and may be a potential target for tumor therapy.

Sacraoxides A-G, Bioactive Cembranoids from Gum Resin of Boswellia sacra.

Seven undescribed cembranoids, sacraoxides A-G (1, 3-8) were isolated from the gum resin of Boswellia sacra. Their structures were elucidated by extensive physicochemical and spectroscopic analysis, as well as ECD calculation, modified Mosher's method and X-ray diffraction crystallography. Compounds 6 and 7 exhibited inhibitory activities on nitric oxide (NO) production induced by lipopolysaccharide in RAW264.7 cells with IC50 values of 24.9 ± 1.7 and 36.4 ± 2.9 µM.

Suppression of S-phase kinase-associated protein 2 induces apoptosis and inhibits tumor growth in human laryngeal cancer.

S-phase kinase-associated protein 2 (Skp2) is related to cellular proliferation and differentiation in laryngeal carcinoma. This study aimed to investigate the effect of Skp-2 suppression on p27 expression, cellular proliferation, apoptosis, and tumor growth in laryngeal squamous cell carcinoma. Skp2 was stably suppressed in Hep2 laryngeal carcinoma cells by lentivirus siRNA technology and Hep2-siRNA was then inoculated into nude mice. The siRNA-induced suppression of Skp2 increased p27 expression, decreased cellular proliferation, and increased apoptosis in human laryngeal carcinoma cells and tumors, indicating that Skp2 is a viable target in the gene therapy treatment of human laryngeal carcinoma.

Overexpression of SATB1 in laryngeal squamous cell carcinoma.

OBJECTIVE: To investigate the expression of special AT-rich sequence-binding protein 1 (SATB1), and the relationship between SATB1 and clinicopathological factors of laryngeal squamous cell carcinoma (LSCC). METHODS: Real-time PCR, Western blot and immunohistochemistry were used to analyze 80 samples of LSCC and 25 samples of control mucosa. The relationship between SATB1 expression and clinicopathological parameters was evaluated. RESULTS: The SATB1 mRNA expression levels in LSCC were 2.5- to 7.5-fold higher than those in control mucosa tissues (p < 0.001). SATB1 protein expression was detected in approximately 66% (53 out of 80) of the LSCC specimens, whereas it was below the detection limit in all the control mucosa specimens (p < 0.001). The SATB1 mRNA levels in positive cervical lymph nodes, in clinical stages III and IV with poor/moderate cell differentiation, were significantly higher than those in negative cervical lymph nodes in clinical stage II with high cell differentiation (p = 0.022, p = 0.005 and p = 0.006, respectively). CONCLUSIONS: SATB1 mRNA and protein levels are elevated in LSCC tissues, and their levels are correlated with clinical stages and differentiation status. The current findings suggest that SATB1 may be a useful marker for the prognosis and assessment of therapeutic effects.

Diagnosis and treatment of lingual thyroglossal duct cyst in newborns.

BACKGROUND: The objective of this study was to explore the diagnosis and treatment method of lingual thyroglossal duct cyst in newborns. METHOD: The clinical data of nine newborns who were diagnosed as lingual thyroglossal duct cyst were retrospectively analyzed. RESULTS: One lingual thyroglossal duct cyst was found when the tongue was pressed with a spatula. The other eight lingual thyroglossal duct cysts were found with a laryngoscope. Three-dimensional computed tomography showed that the cysts were located at the base of tongue, which was round and smooth. Six of nine patients were treated with the puncture method. The fluid was drawn out, and the average volume was 1.4 mL. Follow-up survey lasted for 1 year or more and 33.3% (2/6) of the cases recurred. For the recurrence, the two patients underwent another operation in which most of the cyst walls were removed and they had no recurrence after another year of follow-up survey. Three of the nine patients were treated with the excision method, and they had no recurrence after 1 year of follow-up survey. CONCLUSIONS: The laryngoscope and three-dimensional computed tomography examinations are important for the diagnosis of lingual thyroglossal duct cyst. Surgical removal serves as a radical cure for lingual thyroglossal duct cyst.

Combined antitumor activity of cucurbitacin B and docetaxel in laryngeal cancer.

Combination therapy with multiple drugs is a common practice in the treatment of cancer. The promising clinical activity of docetaxel has promoted considerable interest in combining it with other antitumor agents. To determine whether cucurbitacin B can enhance chemosensitivity to docetaxel in laryngeal cancer, in the present study, we investigated the combined antitumor effect of cucurbitacin B with docetaxel on Hep-2, a human laryngeal cancer cell line. We treated Hep-2 cells with cucurbitacin B alone or in combination with docetaxel and evaluated cell growth, cell cycle distribution, and apoptosis using MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay, flow cytometry, and fluorescent microscopy. Our results showed that, in comparison with single agent treatment, the combination of cucurbitacin B and docetaxel produced greater efficacy in growth inhibition, cell cycle arrest at G2/M phase, and apoptosis induction. Measuring the modulation of regulators in the cell cycle, apoptosis and signal transductions by Western blot analysis showed that the combination effect of cucurbitacin B and docetaxel was due to suppress the expression of p-STAT3 (signal transducers and activators of transcription 3), Bcl-2, and cyclin B1. Moreover, our in vivo studies were reproduced in a mouse xenograft model, where, the combination of cucurbitacin B with docetaxel synergestively inhibited tumor growth. Together, this investigation suggests that cucurbitacin B combined with docetaxel may be a feasible strategy to enhance the effects of chemotherapy in patients with laryngeal cancer.

Analysis of curative effects on laryngeal carcinoma patients in the northeast region of China.

CONCLUSIONS: In the past 20 years, the level of laryngeal carcinoma treatment in our country has been significantly improved. Early diagnosis is the key for increasing the ratio of larynx preservation surgeries and improving survival rates. The main causes of death within 5 years are local recurrence and metastasis. OBJECTIVE: To describe the main treatment methods for laryngeal carcinoma in China in the 1980s and 1990s and their prospective effects and investigate the prognostic factors. PATIENTS AND METHODS: A retrospective investigation was performed on the 1115 laryngeal carcinoma patients receiving treatment in the department of ENT of the First Affiliated Hospital of China Medical University during 1983-1996 and the survival rates and causes of death were analyzed statistically. RESULTS: There were 780 patients surviving for more than 5 years, 260 dead patients, and 75 patients lost to follow-up. According to the cumulative survival rate curve, the 5-year survival rate was 77% (94% for stage I, 89% for stage II, 82% for stage III, and 66% for stage IV). Glottic cancer has the highest 5-year survival rate, followed by supraglottic cancer, subglottic cancer, and transglottic cancer. The 5-year survival rate of patients receiving partial laryngectomy was 85%, while the 5-year survival rate of those receiving total laryngectomy was 68%. The leading causes of death within 5 years were local recurrence and metastasis (70%), and the causes of death were unknown in 14% of cases.

MYO5A inhibition by miR-145 acts as a predictive marker of occult neck lymph node metastasis in human laryngeal squamous cell carcinoma.

INTRODUCTION: Each year, ~50,000 patients worldwide die of laryngeal squamous cell carcinoma (LSCC) because of its highly metastatic properties. However, its pathogenic mechanisms are still unclear, and in particular, the prediction of metastasis remains elusive. This study aimed to define the role of microRNA-145 (miR-145) in LSCC progression. We also aimed to elucidate the clinical significance of the miR-145/MYO5A pathway, especially the predictive function of MYO5A in neck lymph node metastasis. MATERIALS AND METHODS: MYO5A and miR-145 expression was analyzed in 132 patients with LSCC, and associations between their expression and clinicopathological features were evaluated. We validated the regulatory relationship between miR-145b and MYO5A by dual luciferase reporter assay. The role of the miR-145/MYO5A pathway in proliferation, metastasis, and apoptosis was examined in vitro. The predictive functions of MYO5A in neck lymph node metastasis and prognosis were defined according to patient follow-up. RESULTS: Our results showed downregulation of miR-145 in LSCC, which was negatively correlated with MYO5A suppression of LSCC progression and metastasis. MiR-145 directly regulated MYO5A expression in vitro and suppressed LSCC proliferation and invasion while promoting apoptosis by inhibiting MYO5A. CONCLUSION: Notably, overexpression of serum MYO5A in LSCC predicted cervical nodal occult metastasis and poor prognosis, providing an effective indicator for predicting neck lymph node metastasis and assessing LSCC prognosis.

MicroRNA-1469, a p53-responsive microRNA promotes Genistein induced apoptosis by targeting Mcl1 in human laryngeal cancer cells.

Genistein, a plant isoflavone, is reported to have therapeutic potentials in multiple cancers, However, the molecular mechanism underlying promoting cell apoptosis in laryngeal cancer remains unclear. In this study, we report that miR-1469 was induced by genistein in laryngeal cancer. Elevated miR-1469 promoted cell apoptosis and inhibited Mcl1 expression. In addition, we also observed that tumor suppressor p53 was increased under genistein treatment. Elevation of p53 promoted miR-1469 expression, leading to miR-1469 increase and Mcl1 decrease. Therefore, our findings suggest that genistein can suppress laryngeal cancer cell survival through p53 -miR-1469-Mcl1pathway.

Missing diagnosis of neck metastases by routine detecting method in laryngeal carcinomas.

OBJECTIVE: To evaluate the missing diagnosis of neck metastases by routine detecting method (palpation combined with one pathological slide) in laryngeal carcinomas. METHODS: Sixty-six specimens of neck dissections were collected and observed by routine method, transparent method, and continuous sliding method. RESULTS: Totally, 1153 lymph nodes were detected by palpation method and another 1204 lymph nodes were detected by transparent method. The lymph nodes detected by transparent method account for 51.1% of the total, and among them 10 metastases were found, which account for 15.6% (10/64) of metastatic lymph nodes. For those with no metastasis detected by routine method, 50 microm interval continuous sliding method was performed, and 14 tiny metastases were found, which account for 21.9% (14/64) of metastatic lymph nodes. Detecting by routine method, most lymph nodes (95%) were in tumor growth and tumor suffusion stage. The missing diagnosis rate of routine method was 37.5% (24/64). CONCLUSIONS: When routine method was used to detect lymph nodes in neck specimens, missing diagnosis should be considered to select best therapy. Through transparent method small lymph nodes could be found and it is a valuable method to observe pathological changes of small nodes. Continuous sliding method could find micrometastasis precisely, but the work burden is heavy and it is difficult to be widely used.