Transcription factor FoxM1 promotes cyst growth in PKD1 mutant ADPKD.

Autosomal dominant polycystic kidney disease (ADPKD) is driven by mutations in the PKD1 and PKD2 genes, and it is characterized by renal cyst formation, inflammation and fibrosis. Forkhead box protein M1 (FoxM1), a transcription factor of the Forkhead box (Fox) protein super family, has been reported to promote tumor formation, inflammation and fibrosis in many organs. However, the role and mechanism of FoxM1 in regulation of ADPKD progression is still poorly understood. Here, we show that FoxM1 is an important regulator of cyst growth in ADPKD. FoxM1 is upregulated in cyst-lining epithelial cells in Pkd1 mutant mouse kidneys and human ADPKD kidneys. FoxM1 promotes cystic renal epithelial cell proliferation by increasing the expression of Akt and Stat3 and the activation of ERK and Rb. FoxM1 also regulates cystic renal epithelial cell apoptosis through NF-κB signaling pathways. In addition, FoxM1 regulates the recruitment and retention of macrophages in Pkd1 mutant mouse kidneys, a process that is associated with FoxM1-mediated upregulation of monocyte chemotactic protein 1. Targeting FoxM1 with its specific inhibitor, FDI-6, delays cyst growth in rapidly progressing and slowly progressing Pkd1 mutant mouse kidneys. This study suggests that FoxM1 is a central and upstream regulator of ADPKD pathogenesis and provides a rationale for targeting FoxM1 as a therapeutic strategy for ADPKD treatment.

The mechanism and clinical application of farnesyl diphosphate farnesyltransferase 1 in cancer metabolism.

Cancer poses a significant risk to human well-being. Among the crucial characteristics of cancer is metabolic reprogramming. To meet the relentless metabolic needs, cancer cells enhance cholesterol metabolism within the adverse tumor microenvironment. Reprograming cholesterol metabolism includes a series of modifications in the synthesis, absorption, esterification, and metabolites associated with cholesterol. These adjustments have a strong correlation with the proliferation, invasion, metastasis, and other characteristics of malignant tumors. FDFT1, also known as farnesyl diphosphate farnesyltransferase 1, is an enzyme crucial in the process of cholesterol biosynthesis. Its significant involvement in tumor metabolism has garnered considerable interest. The significance of FDFT1 in cancer metabolism cannot be overstated, as it actively interacts with cancer cells. This paper aims to analyze and consolidate the mechanism of FDFT1 in cancer metabolism and explore its clinical application. The goal is to contribute new strategies and targets for the prevention and treatment of cancer metabolism.

Pancreatic ductal adenocarcinoma: CEUS characteristics are correlated with pathological findings and help predict early recurrence after resection.

OBJECTIVES: To identify characteristics of preoperative contrast-enhanced ultrasound (CEUS) that could predict early recurrence after curative resection of pancreatic ductal adenocarcinoma (PDAC). METHODS: From January 2017 to September 2022, a total of 110 patients with PDAC (all confirmed by samples obtained via operation) who underwent CEUS within 1 month before surgery were enrolled. We proposed five CEUS enhancement patterns (Pattern I, homogeneous enhancement; Pattern II, heterogeneous enhancement without cystic components; pattern III, ring enhancement; Pattern IV, starry enhancement; Pattern V, heterogeneous enhancement with cystic components) of PDAC. Clinical-pathologic and CEUS characteristics for predicting early recurrence (recurrence within 1 year after curative resection) were analyzed. Important CEUS characteristics were compared with the pathological findings. RESULTS: Tumor size and TNM stage were closely associated with early recurrence. Incomplete-enhancement and enhancement pattern III, IV and V at CEUS imaging were more prone to early recurrence. Incomplete-enhancement lesions had higher histological tumor grades, less frequent remaining acini, and more frequent necrosis within the tumor. PDACs with pattern I and II had lower histological tumor grades, and pattern III, IV and V had higher histological tumor grades. PDACs with pattern I, II and IV had less frequent intratumoral necrosis than PDACs with pattern III and V, and PDACs with pattern IV had lower MVD values. CONCLUSIONS: PDACs with incomplete enhancement and enhancement pattern III, IV and V were more prone to early recurrence after attempted curative resection, and these important CEUS characteristics were closely related to the pathological findings of PDAC.

Clinical Value of Artificial Intelligence-Based Computer-Aided Diagnosis System Versus Contrast-Enhanced Ultrasound for Differentiation of Benign From Malignant Thyroid Nodules in Different Backgrounds.

OBJECTIVES: The aim of this study was to compare the value of the AI-SONIC ultrasound-assisted diagnosis system versus contrast-enhanced ultrasound (CEUS) for differential diagnosis of thyroid nodules in diffuse and non-diffuse backgrounds. METHODS: A total of 555 thyroid nodules with pathologically confirmed diagnosis were included in this retrospective study. The diagnostic efficacies of AI-SONIC and CEUS for differentiating benign from malignant nodules in diffuse and non-diffuse backgrounds were evaluated, with pathological diagnosis as the gold standard. RESULTS: The agreement between AI-SONIC diagnosis and pathological diagnosis was moderate in diffuse backgrounds (κ = 0.417) and almost perfect in non-diffuse backgrounds (κ = 0.81). The agreement between CEUS diagnosis and pathological diagnosis was substantial in diffuse backgrounds (κ = 0.684) and moderate in non-diffuse backgrounds (κ = 0.407). In diffuse backgrounds, AI-SONIC had slightly higher sensitivity (95.7 vs 89.4%, P = .375), but CEUS had significantly higher specificity (80.0 vs 40.0%, P = .008). In non-diffuse background, AI-SONIC had significantly higher sensitivity (96.2 vs 73.4%, P < .001), specificity (82.9 vs 71.2%, P = .007), and negative predictive value (90.3 vs 53.3%, P < .001). CONCLUSION: In non-diffuse backgrounds, AI-SONIC is superior to CEUS for differentiating malignant from benign thyroid nodules. In diffuse backgrounds, AI-SONIC could be useful for screening of cases to detect suspicious nodules requiring further examination by CEUS.

Differential diagnosis of thyroid nodules by the Demetics ultrasound-assisted diagnosis system and contrast-enhanced ultrasound combined with thyroid image reporting and data systems.

BACKGROUND: More and more new ultrasound techniques with their own characteristics are applied in the differential diagnosis of thyroid nodules. This study amied to assess and compare the diagnostic value of the Demetics ultrasound-assisted diagnosis system and contrast-enhanced ultrasound (CEUS) combined with the Thyroid Image Reporting and Data Systems (TI-RADS) for thyroid nodules. DESIGN AND PATIENTS: A total of 600 thyroid nodules with pathological findings were retrospectively analysed. Demetics and CEUS were performed for all nodules. The diagnostic efficacy of Demetics and CEUS for nodules of different sizes was evaluated and compared in terms of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR+) and negative likelihood ratio (LR-). The characteristics of nodules diagnosed and misdiagnosed by Demetics were compared to analyse the factors affecting the diagnostic accuracy of Demetics. The necessity of CEUS for nodules that are prone to misdiagnosis in Demetics was assessed. RESULTS: Both Demetics and CEUS can be used for the differential diagnosis of benign and malignant thyroid nodules of different sizes. The diagnostic agreement between Demetics and CEUS for thyroid nodules of different sizes was moderate, substantial and fair, respectively. The sensitivity and NPV of Demetics were higher than those of CEUS, and the specificity, PPV and LR+ of CEUS were higher than that of Demetics. The LR- of Demetics was lower than those of CEUS. There were significant differences in age, calcification and margin in analysing the factors affecting Demetics. CEUS correctly diagnosed 50 of the 101 nodules misdiagnosed by Demetics. CONCLUSIONS: Demetics showed high sensitivity in diagnosing thyroid nodules, while CEUS showed high specificity. In clinical practice, CEUS can further improve the diagnostic accuracy for nodules that are easily misdiagnosed by Demetics.

Value of Echogenic Foci in Diagnosing Papillary Thyroid Carcinoma and Predicting Aggressive Biological Behavior.

OBJECTIVES: To assess the diagnostic value of echogenic foci in papillary thyroid carcinoma (PTC) and the relationship between echogenic foci and aggressiveness of PTC. METHODS: From January 2018 to January 2021, a total of 950 patients diagnosed with thyroid nodules (n = 1113) in our hospital were retrospectively analyzed. Among the 1113 nodules, single PTC in 527 patients confirmed by surgery was studied for their aggressive biological behavior. The patterns of echogenic foci were classified as: no echogenic foci, sparse punctate echogenic foci, focal punctate echogenic foci, diffuse punctate echogenic foci, petal-like punctate echogenic foci, comet-tail artifacts, coarse echogenic foci, peripheral rim (eggshell echogenic foci), and mixed echogenic foci. The clinical and ultrasonographic characteristics were also analyzed. A univariate analysis was performed, and binary logistic regression was performed to screen independent risk factors. RESULTS: For the differential diagnosis of PTC, age < 50 years, size <1.1 cm, hypoechoic or very hypoechoic, aspect ratio > 1, irregular shape, types II (punctate echogenic foci) and VI (mixed echogenic foci) were independent risk factors. For the aggressive biological behavior of PTC, male sex, age<42 years, size <1.0 cm, types IIb (focal punctate echogenic foci), IIc (diffuse punctate echogenic foci), and VI (mixed echogenic foci) were independent risk factors for predicting cervical lymph node metastasis of PTC. CONCLUSION: Echogenic foci are useful in diagnosing PTC and predicting aggressiveness of PTC, which contribute to screening invasive PTC and avoiding overdiagnosis and overtreatment.

Identification of hydroxylation at aromatic amino acid residues in yeast kinase using mass spectrometry with affinity enrichment.

RATIONALE: Protein kinases represent the key elements in phosphorylation-based signal transmission. Recent studies suggest that hydroxylation may mediate activities of protein kinases. This paper aims to examine the hydroxylation in protein kinases for improving our understanding of the protein modification. METHODS: We combined affinity-based protein purification with MS analysis for identification of novel hydroxylation at aromatic amino acid residues in yeast kinases. RESULTS: We identified 17 hydroxylation at aromatic amino acid residues (10 at Phe, 1 at Tyr and 6 at Trp) using MS analysis. We further characterized the localization and studied the potential significance of these modifications. CONCLUSIONS: This is a new report on the identification of hydroxylation at aromatic amino acid residues in yeast kinases. This study expands the catalog of hydroxylation in kinases and suggests the potential function of hydroxylation. Copyright © 2016 John Wiley & Sons, Ltd.

Contrast-enhanced ultrasound (CEUS) characteristics of atypical-enhanced papillary thyroid carcinoma (PTC).

OBJECTIVE: To investigate the diagnostic value of CEUS in atypical-enhanced PTC. METHODS: The clinical data, qualitative and quantitative parameters of CEUS in 177 Iso/hyper-enhanced thyroid nodules with definite pathological results were retrospectively analyzed in the Lanzhou University Second Hospital from June 2019 to January 2021. And the clinical value of CEUS in the diagnosis of atypical-enhanced PTC was assessed using univariate and multivariate analysis. RESULTS: Among the 177 thyroid nodules, 59 were benign and 118 were PTC. There were significant differences in age, enhancement border, ring enhancement, speed of wash in, speed of wash out, enhancement pattern, capsule interruption, time to peak, time to wash out, RT, TPH, and TTP (P < 0.05). Multivariate analysis showed unclear enhancement border and concentric enhancement were independent risk factors for the diagnosis of atypical-enhanced PTC by CEUS. The sensitivity, specificity, PPV, NPV, and accuracy of the model in diagnosing atypical-enhanced PTC were 88.1%, 71.2%, 86.0%, 75.0%, and 82.5%, respectively. The area under the curve (AUC) of the receiver operating characteristic (ROC) curve was 0.910. CONCLUSION: The diagnosis of atypical-enhanced PTC can be better performed by enhancement characteristics and time intensity curve (TIC) of CEUS, which have a good clinical application value.

Identification of pyroptosis-related gene signature for predicting prognosis of patients with pancreatic cancer using bioinformatics.

Pancreatic cancer, a common digestive system malignancy, is dubbed the "king of cancers". The role of pyrophosis-related genes (PRGs) in pancreatic cancer prognosis is yet unknown. In pancreatic cancer and normal tissue, we discovered 9 PRGs that are expressed differently in pancreatic cancer and healthy tissue. Based on the differential expression of PRGs, 2 clusters of pancreatic cancer cases could be identified. The 2 groups had significant disparities in total survival time. The prognostic model of a 5-PRGs signature was created using least absolute shrinkage and selection operator (LASSO) method. The median risk score was used to split pancreatic cancer patients in The Cancer Genome Atlas (TCGA) cohort into 2 groups: low risk and high risk. Patients classified as low-risk had significantly higher survival rates than those classified as high-risk (P < .01). The same results were obtained by validating them against the Gene Expression Omnibus database (P = .030). Cox regression statistical analysis showed that risk score was an independent predictor of overall survival in pancreatic cancer patients. Functional enrichment analysis revealed that apoptosis, cell proliferation, and cell cycle-related biological processes and signaling pathways were enriched. Additionally, the immunological status of the high-risk group worsened. In conclusion, a novel pyroptosis-related gene signature can be used to predict pancreatic cancer patient prognosis.

Study on correlation between two-dimensional ultrasound, contrast-enhanced ultrasound and microvascular invasion in hepatocellular carcinoma.

OBJECTIVE: To explore the correlation between two-dimensional ultrasound (2D-US), contrast-enhanced ultrasound (CEUS) and microvascular invasion (MVI) in hepatocellular carcinoma (HCC). METHODS: In this retrospective study, 56 patients with surgically pathologically confirmed HCC lesions were included. Patients were classified according to the presence of MVI: MVI positive group (n = 17) and MVI negative group (n = 39). 2D-US and CEUS examinations were performed within two weeks before surgery. The 2D-US and CEUS features were analyzed for correlation with MVI. Statistically significant parameters of ultrasound characteristic were scored, and the results of the scores were analyzed by ROC curve. RESULTS: There were statistically significant differences in tumor shape, boundary, capsule, CEUS portal phase and delayed phase enhancement pattern, time to wash out, and tumor margin after enhancement (P < 0.05), while there were no statistically significant differences in tumor location and size, CEUS arterial phase enhancement pattern, initial time, time to peak, and peritumor enhancement (P > 0.05). When diagnosing the presence of MVI in HCC patients with cut-off value of the score combined 2D-US and CEUS features≥3, the maximum Jorden index was 0.58, and its diagnostic sensitivity and specificity were 94.10% and 64.1%, respectively, meaning that the total score≥3 was highly suspicious of the presence of MVI. CONCLUSIONS: 2D-US and CEUS are feasible methods for preoperative prediction of MVI in HCC, and can provide some theoretical basis for individualized clinical treatment.

Contrast-Enhanced Ultrasound in the Differential Diagnosis and Risk Stratification of ACR TI-RADS Category 4 and 5 Thyroid Nodules With Non-Hypovascular.

OBJECTIVE: This study aims to investigate the value of contrast-enhanced ultrasound (CEUS) in the differential diagnosis and risk stratification of ACR TI-RADS category 4 and 5 thyroid nodules with non-hypovascular. METHODS: From January 2016 to December 2019 in our hospital, 217 ACR TI-RADS category 4 and 5 nodules with non-hypovascular in 210 consecutive patients were included for a derivation cohort. With surgery and/or fine-needle aspiration (FNA) as a reference, conventional ultrasound (US) features and CEUS features were analyzed. Multivariate logistic regression analysis was used to screen the independent risk factors and establish a risk predictive model. Between January 2020 and March 2021, a second cohort of 100 consecutive patients with 101 nodules were included for an external validation cohort. The model was converted into a simplified risk score and was validated in the validation cohort. The area under the receiver operating characteristic curves (AUC) were used to assess the models' diagnostic performance. RESULTS: Micro-calcification, irregular margin, earlier wash-out, centripetal enhancement, and absence of ring enhancement were independent risk factors and strongly discriminated malignancy in the derivation cohort (AUC = 0.921, 95% CI 0.876-0.953) and the validation cohort (0.900, 0.824-0.951). There was no significant difference (P = 0.3282) between the conventional US and CEUS in differentiating malignant non-hypovascular thyroid nodules, but a combination of them (the predictive model) had better performance than the single method (all P <0.05), with a sensitivity of 87.0%, specificity of 86.2%, and accuracy of 86.6% in the derivation cohort. The risk score based on the independent risk factors divided non-hypovascular thyroid nodules into low-suspicious (0-3 points; malignancy risk <50%) and high-suspicious (4-7 points; malignancy risk ≥ 50%), the latter with nodule ≥10mm was recommended for FNA. The risk score showed a good ability of risk stratification in the validation cohort. Comparing ACR TI-RADS in screening suitable non-hypovascular nodules for FNA, the risk score could avoid 30.8% benign nodules for FNA. CONCLUSIONS: CEUS is helpful in combination with conventional US in differentiating ACR TI-RADS category 4 and 5 nodules with non-hypovascular. The risk score in this study has the potential to improve the diagnosis and risk stratification of non-hypovascular thyroid nodules.

Diagnostic Value of CEUS LI-RADS Version 2017 in Differentiating AFP-Negative Hepatocellular Carcinoma from Other Primary Malignancies of the Liver.

PURPOSE: To explore the diagnostic value of Contrast-enhanced Ultrasound Liver Imaging Reporting and Data System version 2017 (CEUS LI-RADS v2017) in differentiating alpha-fetoprotein (AFP)-negative hepatocellular carcinoma (HCC) from other primary malignancies (OM) of the liver. METHODS: The data of 99 patients with primary liver malignant tumors confirmed by surgical pathology and AFP-negative from January 2018 to January 2021 were retrospectively analyzed, and the lesions were divided into 61 cases in the AFP-negative HCC group and 38 cases in the OM group according to the pathological findings, the CEUS features of the lesions were analyzed and the lesions were classified according to the CEUS LI-RADS v2017. Comparison of CEUS features between the two groups was performed using the χ2 test. The sensitivity, specificity, positive predictive value, negative predictive value, and coincidence rate of CEUS LI-RADS v2017 for the diagnosis of AFP-negative HCC and OM were calculated using pathological findings as the gold standard. RESULTS: The differences in features of arterial phase enhancement and wash-out between the HCC and OM groups were statistically significant (p < 0.05). The sensitivity of diagnosing HCC by LR-5 was 62.3% and the specificity was 92.1%. The sensitivity of diagnosing OM by LR-M was 92.1% and the specificity was 83.6%. CONCLUSIONS: When AFP is negative in patients with intrahepatic focal lesions, LR-5 has high specificity but low sensitivity in the diagnosis of HCC, and LR-M has high sensitivity and specificity in the diagnosis of OM. CEUS LI-RADS is a tool to differentiate AFP-negative HCC and OM effectively.

Value of Combining Clinical Factors, Conventional Ultrasound, and Contrast-Enhanced Ultrasound Features in Preoperative Prediction of Central Lymph Node Metastases of Different Sized Papillary Thyroid Carcinomas.

PURPOSE: Early and accurate preoperative diagnosis of central lymph node metastasis (CLNM) is crucial to improve surgical management of patients with clinical lymph node-negative papillary thyroid carcinoma (PTC). Towards improving diagnosis of CLNM, we assessed the value of combining preoperative clinical characteristics, conventional ultrasound, and contrast-enhanced ultrasound (CEUS) in preoperative prediction of CLNM of different sized PTCs. PATIENTS AND METHODS: Patients were divided according to tumor size: a PTC group (>10 mm) and a papillary thyroid microcarcinoma (PTMC) group (≤10 mm). We retrospectively analyzed the clinical and ultrasonographic features of 120 PTC patients and 165 PTMC patients. Multivariate logistic regression analysis was used to screen independent risk factors and establish prediction models. Receiver operating characteristic curves were used to determine the best cut-off values for continuous variables and assess the performance of prediction models. RESULTS: Independent risk predictors of CLNM for the PTC group were extrathyroidal extension in CEUS (OR=7.923), tumor size >14 mm (OR=5.491), and multifocality (OR=3.235). For the PTMC group, the independent risk factors were the distance from the thyroid capsule =0 mm (OR=4.629), male (OR=3.315), tumor size >5 mm (OR=3.304), and microcalcification (OR=2.560). The predictive model of combined method had better performance in predicting CLNM of PTC compared with models based on CEUS and conventional ultrasound alone (area under the curve: 0.832 vs 0.739, P=0.0011; 0.832 vs 0.678, P=0.0012). For PTMC, comparing with CEUS, the combined method and conventional ultrasound performed better than CEUS alone in predicting CLNM (area under the curve: 0.783 vs 0.636, P=0.0016; 0.738 vs 0.636, P=0.0196). CONCLUSION: The predictive models of combined method obtained from significant preoperative clinical and ultrasonographic features can potentially improve the preoperative diagnosis and individual treatment of CLNM in patients with PTC and PTMC. CEUS may be helpful in predicting CLNM of PTC, but CEUS would be ineffective in predicting CLNM of PTMC.

Comparative analysis of histone H3 and H4 post-translational modifications of esophageal squamous cell carcinoma with different invasive capabilities.

Eukaryotic DNA is packaged into a chromatin with the help of four core histones (H2A, H2B, H3, and H4). Diverse histone post-translational modifications (PTMs) are hence involved in the regulation of gene transcription. However, how this regulation does work is still poorly understood and lacks details. Here we used the mass spectrometry-based proteomics approach to perform a comparative analysis of histone marks at a global level in two phenotypes of esophageal squamous cell carcinoma (ESCC) with different invasiveness. We obtained a comprehensive profiling of histone H3 and H4 PTMs including lysine methylation, acetylation and novel butyrylation. The correlation between histone marks and cancer invasive capabilities was further characterized and one distinguishable PTM, H4K79me2 was discovered and verified in this study. Immunohistochemistry analysis suggests that abnormal level of H4K79me2 may be related to poor survival of ESCC patients. Our results enrich the dataset of the feature pattern of global histone PTMs in ESCC cell lines. BIOLOGICAL SIGNIFICANCE: Core histone proteins, decorated by multiple biological significant protein post-translational modifications (PTMs) such as lysine acetylation and lysine methylation, are considered to regulate gene transcription and be associated with the development of cancer. Recent studies have further shown that global level of histone modifications is the potential hallmark of cancer to predict the clinical outcomes of human cancers. However, the regulation mechanism is largely unknown. Here we used the mass spectrometry based proteomics coupled with stable isotope labeling with amino acids in cell culture (SILAC) to characterize the global levels of histone marks in two phenotypes of esophageal squamous cell carcinoma (ESCC) cell lines with different invasive capabilities. To the best of our knowledge, it is the first report about the comparative analysis for histone marks of the different invasive ESCC cell lines. A significantly differential level of histone modification, H4K79me2, was determined and verified. Immunohistochemistry analysis further suggests that abnormal level of H4K79me2 may be related to poor survival of ESCC patients. Our results could contribute to understanding the different expressions of global histone PTMs in different invasive ESCC cell lines.

[Identification of methylation at lysine 27 and 36 sites in histone H3].

We combined high performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) with bioinformatics tools to analyze the isobaric modified peptides which were methylated and dimethylated at either lysine (K) 27 or/and K36 from histone H3. They were identified and dissected through alignment of every fragment ion, and the two modified sites were further analyzed according to their relative intensities of MS/MS spectra.

Effects of Xinjining extract on inward rectifier potassium current in ventricular myocytes of guinea pig.

OBJECTIVE: To study the effect of Xinjining extract (, XJN) on inward rectifier potassium current (I(K1)) in ventricular myocyte (VMC) of guinea pigs and its anti-arrhythmic mechanism on ion channel level. METHODS: Single VMC was enzymatically isolated by zymolisis, and whole-cell patch clamp recording technique was used to record the I(k1) in VMC irrigated with XJN of different concentrations (1.25, 2.50, 5.00 g/L; six samples for each). The stable current and conductance of the inward component of I(K1) as well as the outward component of peak I(K1) and conductance of it accordingly was recorded when the test voltage was set on -110 mV. RESULTS: The suppressive rate of XJN on the inward component of I(K1) was 9.54% + or - 5.81%, 34.82% + or - 15.03%, and 59.52% + or - 25.58% with a concentration of 1.25, 2.50, and 5.00 g/L, respectively, and that for the outward component of peak I(K1) was 23.94% + or - 7.45%, 52.98% + or - 19.62%, and 71.42% + or - 23.01%, respectively (all P<0.05). Moreover, different concentrations of XJN also showed effects for reducing I(K1) conductance. CONCLUSION: XJN has inhibitory effect on I(K1) in guinea pig's VMC, and that of the same concentration shows stronger inhibition on outward component than on inward component, which may be one of the mechanisms of its anti-arrhythmic effect.