F2RL3 Regulates Epithelial-Mesenchymal Transition and Angiogenesis in Gastric Cancer through the Rap1/MAPK Signaling Pathway.

BACKGROUND: Gastric cancer (GC) is frequently diagnosed at advanced stages, when cancer cells have already metastasized. Therefore, patients with GC have a low survival rate and poor prognosis even after treatment. METHODS: We downloaded GC-related RNA sequencing (RNA-Seq) data, copy number variation (CNV) data, and clinical data for bioinformatics analysis to screen prognostic genes of GC. Single-sample gene set enrichment analysis and survival analyses were performed on the RNA-Seq data, and differential and correlation analyses were conducted on the CNV data to obtain CNV-driven differentially expressed genes (DEGs). Prognostic genes were identified through univariate Cox analyses of the CNV-driven DEGs, combined with the clinical data. F2R like thrombin or trypsin receptor 3 (F2RL3) was finally selected for verification after functional and survival analyses of the prognostic genes. RESULTS: F2RL3 expression was lower in paracancer tissue than in GC tissue, and lower in GES-1 gastric epithelial cells than in GC cells. The cell culture supernatants from F2RL3-knockdown GC cells were collected and used to culture human umbilical vein endothelial cells (HUVECs). It was observed that F2RL3 enhanced the activity, metastasis, invasion, and angiogenesis of GC cells; promoted the epithelial-mesenchymal transition (EMT) of GC cells; and impacted the Ras-associated protein 1 (Rap1)/mitogen-activated protein kinase (MAPK) pathway. To further explore the involvement of the Rap1/MAPK pathway in GC development, a pathway activator was added to GC cells with knockdown of F2RL3 expression. This pathway activator not only enhanced the activity, invasion, and migration of GC cells but also promoted the EMT and blood vessel formation. CONCLUSIONS: F2RL3 regulates the angiogenesis and EMT of GC cells through the Rap1/MAPK pathway, thus influencing the onset and progression of GC.

TRIM21-Promoted FSP1 Plasma Membrane Translocation Confers Ferroptosis Resistance in Human Cancers.

Ferroptosis, an iron-dependent form of regulated cell death driven by excessive accumulation of lipid peroxides, has become a promising strategy in cancer treatment. Cancer cells exploit antioxidant proteins, including Ferroptosis Suppressor Protein 1 (FSP1), to prevent ferroptosis. In this study, it is found that the E3 ubiquitin ligase TRIM21 bound to FSP1 and mediated its ubiquitination on K322 and K366 residues via K63 linkage, which is essential for its membrane translocation and ferroptosis suppression ability. It is further verified the protective role of the TRIM21-FSP1 axis in RSL3-induced ferroptosis in cancer cells and a subcutaneous tumor model. Moreover, TRIM21 is highly expressed in multiple gastrointestinal (GI) tumors, and its expression is further stimulated upon ferroptosis induction in cancer cells and the KPC mouse model. In summary, This study identifies TRIM21 as a negative regulator of ferroptosis through K63 ubiquitination of FSP1, which can serve as a therapeutic target to enhance the chemosensitivity of tumors based on ferroptosis induction.

FGFR2 upregulates PAI-1 via JAK2/STAT3 signaling to induce M2 polarization of macrophages in colorectal cancer.

Fibroblast growth factor receptor 2 (FGFR2) is frequently activated by overexpression or mutation, and an abnormal fibroblast growth factor (FGF)/FGFR signaling pathway is associated with the occurrence, development, and poor prognosis of colorectal cancer (CRC). Our preliminary analysis found that plasminogen activator inhibitor-1 (PAI-1) expression may be related to FGF/FGFR signaling, however, their role in the tumor immune microenvironment remains unclear. In this study, we observed markedly higher PAI-1 expression in CRC patients with poor survival rates. PAI-1 is regulated by FGF/FGFR2 in colon cancer cells and is involved in M2 macrophage polarization. Mechanistically, inhibiting the JAK2/STAT3 signaling pathway could cause PAI-1 downregulation. Furthermore, the activation of phosphorylated STAT3 upregulated PAI-1. In vivo, FGFR2 overexpression in tumor-bearing mouse models suggested that a PAI-1 inhibitor could rescue FGFR2/PAI-1 axis-induced M2 macrophage polarization, which leads to effective immune activity and tumor suppression. Moreover, the combination of a PAI-1 inhibitor and anti-PD-1 therapy exhibited superior antitumor activity in mice. These findings offer novel insights into the molecular mechanisms underlying tumor deterioration and provide potential therapeutic targets for CRC treatment.

A self-amplifying USP14-TAZ loop drives the progression and liver metastasis of pancreatic ductal adenocarcinoma.

With a 5-year survival rate of approximately 10%, pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal solid malignancies in humans. A poor understanding of the underlying biology has resulted in a lack of effective targeted therapeutic strategies. Tissue microarray and bioinformatics analyses have revealed that the downstream transcriptional coactivator of the Hippo pathway, transcriptional coactivator with PDZ-binding motif (TAZ), might be a therapeutic target in PDAC. Since pharmacological inhibition of TAZ is challenging, we performed unbiased deubiquitinase (DUB) library screening to explore the pivotal regulators of TAZ ubiquitination as potential targets in PDAC models. We found that USP14 contributed to Yes-associated protein (YAP)/TAZ transcriptional activity and stabilized TAZ but not YAP. Mechanistically, USP14 catalyzed the K48-linked deubiquitination of TAZ to promote TAZ stabilization. Moreover, TAZ facilitated the transcription of USP14 by binding to the TEA domain transcription factor (TEAD) 1/4 response element in the promoter of USP14. USP14 was found to modulate the expression of TAZ downstream target genes through a feedback mechanism and ultimately promoted cancer progression and liver metastasis in PDAC models in vitro and in vivo. In addition, depletion of USP14 led to proteasome-dependent degradation of TAZ and ultimately arrested PDAC tumour growth and liver metastasis. A strong positive correlation between USP14 and TAZ expression was also detected in PDAC patients. The small molecule inhibitor of USP14 catalytic activity, IU1, inhibited the development of PDAC in subcutaneous xenograft and liver metastasis models. Overall, our data strongly suggested that the self-amplifying USP14-TAZ loop was a previously unrecognized mechanism causing upregulated TAZ expression, and identified USP14 as a viable therapeutic target in PDAC.

Inhibition of BRD4 enhanced the tumor suppression effect of dasatinib in gastric cancer.

BRD4, a member of the bromodomain and extraterminal (BET) family, is elevated in multiple cancer tissues, including gastric cancer (GC). Targeted therapy with BRD4 may help improve the overall survival of patients with GC. Meanwhile, the approved multi-target kinase inhibitor, dasatinib, was recently reported to show varied tumor-suppressive effects in GC cells. This study investigated BRD4 expression in vivo and in vitro using immunohistochemistry and western blotting, respectively. We discussed the relationship between BRD4 expression and patient prognosis. Next, the antitumor efficacy of dasatinib was measured in BRD4-knockdown GC cells to determine the role of BRD4 blockage in dasatinib treatment. Finally, molibresib, a BET inhibitor, was used to measure the cooperative function of BRD4 inhibition and dasatinib treatment in three GC cell lines. Epithelial BRD4 expression was higher in tumoral and metastatic tissues and was strongly associated with unfavorable tumor, node, and metastasis stages and survival. BRD4 expression was heterogeneous in the three GC cell lines tested in vitro. In SGC7901, a BRD4-high GC cell line, knockdown of BRD4 using specific siRNAs suppressed cell growth individually and cooperatively with dasatinib. Moreover, molibresib and dasatinib showed a cooperative effect in suppressing the proliferation of BRD4-high GC cells. In conclusion, we confirmed that increased epithelial BRD4 expression is associated with poor disease stage and prognosis in GC and BRD4 blockage might be a valuable strategy to improve the sensitivity of dasatinib and other drugs in the chemotherapy of advanced GC.

Characteristics of gut microbiota in patients with gastric cancer by surgery, chemotherapy and lymph node metastasis

Gastric cancer (GC) is a malignant gastrointestinal tumor that can result in high mortality. Surgery and chemotherapy are often used for the effective treatment of GC. In addition, lymph node metastasis is a significant factor affecting the therapy of GC. Current researches have revealed that gut microbiota has the potential as biomarkers to distinguish healthy people and GC patients. However, the relationship between surgery, chemotherapy, and lymph node metastasis is still unclear.MethodsIn this study, 16S rRNA sequencing was used to investigate 157 GC fecal samples to identify the role of surgery, chemotherapy, and lymph node metastasis. Immunohistochemical analysis was used to value the expression of Ki67, HER2 in GC patient tissues.ResultsThere exist some gut microbiotas which can distinguish surgery from non-surgery GC patients, including Enterococcus, Megasphaera, Corynebacterium, Roseburia, and Lachnospira. Differences between lymph node metastasis and chemotherapy in GC patients are not significant. Moreover, we found the abundance of Blautia, Ruminococcus, Oscillospira were related to the expression of Ki67 and the abundance of Prevotella, Lachnospira, Eubacterium, Desulfovibiro were correlated with the expression of HER2.ConclusionsThe choice of treatment has a certain impact on the intestinal flora of patients with gastric cancer. Our research shows that surgery has a great effect on the intestinal flora of patients with gastric cancer. However, there were no significant differences in the characteristics of intestinal flora in patients with gastric cancer whether they received chemotherapy or whether they had lymph node metastasis. In addition, the association of gut microbiota with Ki67 and HER2 indicators is expected to provide the possibility of gut microbiota as a tumor prognostic marker. (AU)

LncRNA FAM83H-AS1 promotes the malignant progression of pancreatic ductal adenocarcinoma by stabilizing FAM83H mRNA to protect ß-catenin from degradation.

BACKGROUND: Pancreatic ductal adenocarcinoma is prone to metastasis, resulting in short survival and low quality of life. LncRNAs are pivotal orchestrators that participate in various tumor progress. The underlying role and mechanism of lncRNA FAM83H-AS1 is still unknown in PDAC progression. METHODS: To address this issue, firstly, we profiled and analyzed the aberrant lncRNA expression in TCGA database and identified FAM83H-AS1 as the most effective one in promoting the migration of pancreatic cancer cells. Then, the expression levels of FAM83H-AS1 in patient's serum, tumor tissues and PDAC cells were detected using RT-qPCR, and FAM83H-AS1 distribution in PDAC cells was determined by performing FISH and RT-qPCR. Next, a series of in vivo and in vitro functional assays were conducted to elucidate the role of FAM83H-AS1 in cell growth and metastasis in PDAC. The regulatory relationship between FAM83H-AS1 and FAM83H (the homologous gene of FAM83H-AS1) was verified by performing protein and RNA degradation assays respectively. Co-IP assays were performed to explore the potential regulatory mechanism of FAM83H to ß-catenin. Rescue assays were performed to validate the regulation of the FAM83H-AS1/FAM83H/ß-catenin axis in PDAC progression. RESULTS: FAM83H-AS1 was highly expressed in the tumor tissues and serum of patients with PDAC, and was correlated with shorter survival. FAM83H-AS1 significantly promoted the proliferation, invasion and metastasis of PDAC cells, by protecting FAM83H mRNA from degradation. Importantly, FAM83H protein manifested the similar malignant functions as that of FAM83H-AS1 in PDAC cells, and could bind to ß-catenin. Specifically, FAM83H could decrease the ubiquitylation of ß-catenin, and accordingly activated the effector genes of Wnt/ß-catenin signaling. CONCLUSIONS: Collectively, FAM83H-AS1 could promote FAM83H expression by stabilizing its mRNA, allowing FAM83H to decrease the ubiquitylation of ß-catenin, thus resulted in an amplified FAM83H-AS1/FAM83H/ß-catenin signal axis to promote PDAC progression. FAM83H-AS1 might be a novel prognostic and therapeutic target for combating PDAC.

Damage Identification of Semi-Rigid Joints in Frame Structures Based on Additional Virtual Mass Method.

In civil engineering, the joints of structures are complex, and their damage is generally hard to be detected. Due to the insensitivity of structural modal information to local joint damage, this paper presents a method based on additional virtual mass for damage identification of a semi-rigid joint in a frame structure. Firstly, the modeling of a semi-rigid is described. Secondly, the frequency response of the virtual structure is constructed, and the natural frequency of the constructed virtual structure is extracted by the ERA method. By adding multiple values of virtual masses at different positions, the natural frequency information sensitive to joint damage for damage identification is effectively increased. Based on the above theory, qualitative identification of joint damage is proposed to detect the potential damage, and identification of both damage location and its extent is presented, using natural frequency. Improved Orthogonal Matching Pursuit (IOMP) algorithm is employed to improve the accuracy of the natural frequency-based method for damage identification. At last, numerical simulation of a three-story frame is performed to discuss and to verify the effectiveness of the proposed method.

Characteristics of gastric cancer gut microbiome according to tumor stage and age segmentation.

With the development of 16S rRNA technology, gut microbiome evaluation has been performed in many diseases, including gastrointestinal tumors. Among these cancers, gastric cancer (GC) exhibits high morbidity and mortality and has been extensively studied in its pathogenesis and diagnosis techniques. The current researches have proved that the gut microbiome may have the potential to distinguish GC patients from healthy patients. However, the change of the gut microbiome according to tumor node metastasis classification (TNM) has not been clarified. Besides, the characteristics of gut microbiome in GC patients and their ages of onset are also ambiguous. To address the above shortcomings, we investigated 226 fecal samples and divided them according to their tumor stage and onset age. The findings revealed that surgery and tumor stage can change the characteristic of GC patients' gut microbiota. In specific, the effect of surgery on early gastric cancer (EGC) was greater than that on advanced gastric cancer (AGC), and the comparison of postoperative microflora with healthy people indicated that EGC has more differential bacteria than AGC. Besides, we found that Collinsella, Blautia, Anaerostipes, Dorea, and Lachnospiraceae_ND3007_group expressed differently between EGC and AGC. More importantly, it is the first time revealed that the composition of gut microbiota in GC is different between different onset ages. KEY POINTS: •Gut microbiota of gastric cancer (GC) patients are either highly associated with TNM stage and surgery or not. It shows surgery has more significant changes in early gastric cancer (EGC) than advanced gastric cancer (AGC). •There existed specific gut microbiota between EGC and AGC which may have potential to distinguish the early or advanced GC. •Onset age of GC may influence the gut microbiota: the composition of gut microbiota of early-onset gastric cancer (EOGC) and late-onset gastric cancer (LOGC) is significantly different.

Cultural and Creative Product Design and Image Recognition Based on the Convolutional Neural Network Model.

The development in technology has resulted in the utilization of artificial intelligence systems in various fields. In this research, we are going to study cultural and creative product design and image recognition based on a convolutional neural network (CNN) model. A convolutional neural network is referred to as a type of artificial neural network (ANN) that is used to analyze visual images. Our proposed system deploys a convolutional neural network model for image recognition in the field of cultural and creative product design. Cultural and creative products are becoming more popular these days. The cultural and creative products are referred to as innovative products or innovative new product design which makes use of the cultural symbols and other cultural factors in their design. In simple words, it is the integration of culture and creativity in a new product design. The main aspect of cultural creative products is the incorporation of cultural features into a new product, thus obtaining a creative- and culture-based product. The study results have proved that CNN has provided an accuracy of 87%.

Characteristics of gut microbiota in patients with gastric cancer by surgery, chemotherapy and lymph node metastasis.

BACKGROUND: Gastric cancer (GC) is a malignant gastrointestinal tumor that can result in high mortality. Surgery and chemotherapy are often used for the effective treatment of GC. In addition, lymph node metastasis is a significant factor affecting the therapy of GC. Current researches have revealed that gut microbiota has the potential as biomarkers to distinguish healthy people and GC patients. However, the relationship between surgery, chemotherapy, and lymph node metastasis is still unclear. METHODS: In this study, 16S rRNA sequencing was used to investigate 157 GC fecal samples to identify the role of surgery, chemotherapy, and lymph node metastasis. Immunohistochemical analysis was used to value the expression of Ki67, HER2 in GC patient tissues. RESULTS: There exist some gut microbiotas which can distinguish surgery from non-surgery GC patients, including Enterococcus, Megasphaera, Corynebacterium, Roseburia, and Lachnospira. Differences between lymph node metastasis and chemotherapy in GC patients are not significant. Moreover, we found the abundance of Blautia, Ruminococcus, Oscillospira were related to the expression of Ki67 and the abundance of Prevotella, Lachnospira, Eubacterium, Desulfovibiro were correlated with the expression of HER2. CONCLUSIONS: The choice of treatment has a certain impact on the intestinal flora of patients with gastric cancer. Our research shows that surgery has a great effect on the intestinal flora of patients with gastric cancer. However, there were no significant differences in the characteristics of intestinal flora in patients with gastric cancer whether they received chemotherapy or whether they had lymph node metastasis. In addition, the association of gut microbiota with Ki67 and HER2 indicators is expected to provide the possibility of gut microbiota as a tumor prognostic marker.

Hsa_circ_0007967 promotes gastric cancer proliferation through the miR-411-5p/MAML3 axis.

Circular RNAs are an important kind of noncoding RNAs and involved in cancerogenesis, but the specific mechanism between gastric cancer and circRNAs needs further study. Hsa_circ_0007967 was selected by RNA sequencing. Here, hsa_circ_0007967 was highly expressed in gastric cancer tissues than adjacent normal tissues. Overexpressing hsa_circ_0007967 promoted gastric cancer cell proliferation in vitro and in vivo, while suppression of hsa_circ_0007967 inhibited gastric cancer cell proliferation in vitro and in vivo. Mechanistically, hsa_circ_0007967 sponged miR-411-5p to increase MAML3 expression. Overall, hsa_circ_0007967 is a promising biomarker for gastric cancer diagnosis and a potential molecule for gastric cancer treatment.

Preparation and Study of Folate Modified Albumin Targeting Microspheres.

In this study, folate modified bovine serum albumin was successfully synthesized, while preparation of Nintedanib albumin microspheres (ND-FSA NPs) as a carrier was carried out via electrospinning technology. Folate modified albumin was used to enhance the targeting potential of the prepared microspheres. The prepared microspheres had spherical appearance and smooth outer surface. The diameters of microspheres (764.68 ± 88.46 nm) and zeta potential (- 18.38 ± 0.41 mV) were acceptable. The prepared ND-FSA NPs demonstrated a good degree of modification, wherein the modification rate was 28.1%. In vitro release was significantly increased in three different media (double deionized water-DDW, HCl-pH 1.2, and phosphate buffered solution containing 0.5% Tween 80). It is worth noting that incorporation of Nintedanib into folic acid modified albumin microspheres resulted in an enhanced uptake of the drug into MCF-7 breast cancer cells coupled with higher inhibition rate. Altogether, incorporation of Nintedanib into folate modified albumin microspheres is a new approach to improve water solubility and targeting effect of the drug.

Konjac glucomannan enhances 5-FU-induced cytotoxicity of hepatocellular carcinoma cells via TLR4/PERK/CHOP signaling to induce endoplasmic reticulum stress.

5-Fluorouracil (5-FU) is a commonly used chemotherapeutic agent for various cancers. However, the drug resistance developed by tumor cells hinders the therapeutic effect. Konjac glucomannan (KGM) is indicated to sensitize 5-FU-resistant hepatocellular carcinoma (HCC) cells to 5-FU. In our study, we found that KGM or 5-FU treatment alone did not affect the malignant cell behaviors and endoplasmic reticulum (ER) stress of 5-FU-resistant HCC cells or HepG2/5-FU and Bel-7402/5-FU cells, while cotreatment with KGM and 5-FU significantly facilitated HCC cell apoptosis and ER stress and suppressed cell proliferation potential and migration abilities. Moreover, we explored the underlying mechanism by which KGM induces 5-FU cytotoxicity in HCC cells. We found that Toll-like receptor 4 (TLR4) was downregulated in KGM- and 5-FU-treated HCC cells. TLR4 overexpression reversed the KGM and 5-FU cotreatment-induced inhibition of the malignant behaviors of 5-FU-resistant HCC cells. Furthermore, KGM enhanced 5-FU-induced ER stress by inhibiting TLR4 to activate PERK/ATF4/CHOP signaling. Xenograft mouse models were established using HepG2/5-FU cells, and KGM was demonstrated to reverse 5-FU resistance in HCC tumors in vivo by suppressing TLR4 to enhance ER stress and activate PERK/ATF4/CHOP signaling. In conclusion, KGM combined with 5-FU treatment significantly promoted apoptosis and reduced cell proliferation, migration and ER stress in 5-FU-resistant HCC cells compared with KGM or 5-FU treatment alone by downregulating TLR4 to activate PERK/ATF4/CHOP signaling.

Pyroptosis: A new frontier in cancer.

Pyroptosis is an inflammatory form of cell death triggered by certain inflammasomes, leading to the cleavage of gasdermin D (GSDMD) and activation of inactive cytokines like IL-18 and IL-1ß. Pyroptosis has been reported to be closely associated to some diseases like atherosclerosis and diabetic nephropathy. Recently, some studies found that pyroptosis can influence the proliferation, invasion and metastasis of tumor, which regulated by some non-coding RNAs and other molecules. Hence, we provided an overview of morphological and molecular characteristics of pyroptosis. We also focus on mechanism of regulating pyroptosis in tumor cells as well as the potential roles of pyroptosis in cancer to explore potential diagnostic markers in cancers contributing to the prevention and treatment in cancers.

MiR-139-5p suppresses osteosarcoma cell growth and invasion through regulating DNMT1.

BACKGROUND: Accumulating evidence has suggested the crucial roles of differentially expressed miRNAs in osteosarcoma progression. MiR-139-5p was decreased in various cancers. However, the role of miR-139-5p in the development of osteosarcoma and the underlying mechanism remain to be addressed. METHODS: MiR-139-5p and DNA methyltransferase-1 (DNMT1) mRNA expressions in osteosarcoma tissues and cells were detected by qRT-PCR and western blot analysis. The effects of miR-139-5p and DNMT1 on osteosarcoma cell migration, invasion and epithelial-mesenchymal transition (EMT) were investigated through cell migration and invasion assays, and western blot analysis. The relationship between miR-139-5p and DNMT1was explored using luciferase reporter analysis and western blot. A xenograft tumor model was employed to verify the effects of miR-139-5p on osteosarcoma. RESULTS: We found that miR-139-5p was strikingly decreased in osteosarcoma tissues and cell lines. MiR-139-5p over-expression suppressed osteosarcoma cell growth, migration and invasion, while loss of miR-139-5p promoted osteosarcoma cell proliferation, migration and invasion. Following, we characterized that DNMT1 was a direct target of miR-139-5p that interacted with the 3'-untranslated region of DNMT1. MiR-139-5p regulated a down-regulation in DNMT1 protein expression levels. We also found that DNMT1 expression was increased and negatively correlated with miR-139-5p expression in osteosarcoma tissues clinically. Xenograft tumor analysis suggested that miR-139-5p over-expression reduced tumor growth in osteosarcoma in vivo through decreasing DNMT1 expressions. CONCLUSION: MiR-139-5p suppressed the osteosarcoma progression by reducing DNMT1, supplying new insight into the molecular mechanism uncovering osteosarcoma growth.

[Bacterial culture and drug sensitivity analysis of upper urinary tract calculi complicating with infection].

OBJECTIVE: To investigate the bacteriology and drug sensitivity of upper urinary tract calculi patients, and to provide information for choosing suitable antibiotics. METHODS: In the study, 21 patients who suffered from lithiasis in upper urinary tract and required an emergency drainage for acute obstruction and infection were the "acute group"; 64 patients with calculi in upper urinary tract and accompanied with no infectious symptoms were the "common group". The bacteriology and drug sensitivity of the two groups were investigated. RESULTS: Gram-negative bacteria infected the most common of upper urinary tract calculi patients with infection, accounting for 71.4% in the acute group and 65.7% in the common group, among which Escherichia coli were the predominant ones (35.7% in the acute group and 32.9% in the common group). No difference was found between these two groups in bacterial distribution (P>0.05). Although the average drug resistance rate of Gram-negative bacteria in the acute group was higher than that in the common group, it revealed no significant difference (P>0.05). The drug resistance rate to semisynthetic penicillin, cefuroxime and ceftriaxone were more than 50%, 60%, and 50%, respectively. Quinolones, such as ciprofloxacin and levofloxacin, got a 45% drug resistance. Aminoglycoside, carbapenema were sensitive to Gram-negative bacteria. Cefoperazone/sulbactam and piperacillin/tazobactam were more effective than ceftriaxone and piperacillin, respectively. CONCLUSION: There was no significant difference between upper urinary tract calculi patients with acute infection and common infection in bacteriology and drug sensitivity. Semisynthetic penicillin, the second generation of cephalosporin and quinolone were no longer the good choices of empirical use. Antibiotics combined with ß-lactamase inhibitors would be an ideal empirical therapeutic choice.