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The task of this article is to harness the wisdom of a crowd without calibration. We assume each expert to form predictions by linearly combining various information cues inspired by the lens model, and use Gaussian process to account for sampling and judgmental errors in quantile judgments. Without knowing the experts' observed information cues, we develop a three-step estimation algorithm to factor quantile judgments into "variable profiles" (latent cues underlying each variable of interest) and "expert profiles" (each expert's weights over these cues). We can inquire about expert similarity using their weights of the latent cues, which preserve the same clustering results as the actual weights of the observed cues up to a full-rank linear transform. We can then depict the diversity and dependency among experts explicitly and retain a subcrowd by picking delegates from each subgroup of experts based on the estimated weights. Simulation and case studies demonstrate that a subcrowd selected this way can represent the entire expert panel well. [ABSTRACT FROM AUTHOR]

Gait stability in exercise is an inevitable and vexing problem in mechanics, artificial intelligence, sports, and rehabilitation medicine research. With the rapid development and popularization of science and technology, it becomes a reality for researchers to obtain large-scale human motion data sets in real time with higher efficiency. However, at present, the analysis of gait stability of moving joints is still based on image recognition technology, which is ten times less accurate and inefficient. In this paper, Vicon 3D motion capture system, dynamometer, and surface electromyography system were used to obtain the parameters of the lower limbs of the subjects. Using Anywhere modeling and simulation system, simulation experiments were carried out, and the reaction force data of lower limb joints under two environments were obtained. The gait characteristics of human gait were analyzed from the angle of internal and external adjustment mechanism. Combining one-way ANOVA and incremental occupancy rate, the adjustment process of gait stability is described comprehensively. The findings of this study can provide a theoretical basis for the research of lower limb con-assistive devices and can guide the design and development of bipedal anthropomorphic robots. [ABSTRACT FROM AUTHOR]

Objectives: The tumor mutational burden (TMB) is closely related to immunotherapy outcome. However, the cost of TMB detection is extremely high, which limits its use in clinical practice. A new indicator of genomic instability, the average copy number variation (CNVA), calculates the changes of 0.5‐Mb chromosomal fragments and requires extremely low sequencing depth. Methods: In this study, 50 samples (23 of which were from patients who received immunotherapy) were subjected to low‐depth (10X) chromosome sequencing on the MGI platform. CNVA was calculated by the formula avg (abs (copy number‐2)). In addition, CNVA and TMB were compared with regard to their ability to predict immune infiltration in 509 patients from TCGA. Results: The high‐CNVA group had higher expression levels of PD‐L1, CD39 and CD19 and a higher degree of infiltration of CD8+ T cells and CD3 + T cells. Among the 23 patients treated with immunotherapy, the average CNVA value of the stable disease/partial response group was higher than that of the progressive disease group (P < 0.05). Whole‐genome sequencing data of 509 patients from TCGA and RT‐PCR results of 22 frozen specimens showed that CNVA is more effective than TMB in indicating infiltration of CD8+ T cells and expression of PD‐L1, and CNVA also showed a specific positive correlation with TMB (r = 0.2728, P < 0.0001). Conclusions: Copy number variation can be a good indicator of immune infiltration and immunotherapy efficacy, and with its low cost, it is expected to become a substitute for TMB. [ABSTRACT FROM AUTHOR]

Background: Many tumor‐derived endothelial cells (TECs) are shed into the blood and turn into circulating TECs (CTECs). Rare circulating non‐hematologic aneuploid cells contain CTCs and CTECs, which are biologically and functionally different from each other. CD31 is one of the most representative endothelial cell (EC) markers, yet CD31 alone is not sufficient to detect malignant CTECs due to the existence of abundant normal ECs in circulation. Aneuploidy of chromosome 8 (CEP8) is an important criterion for the identification of malignant cells. Combined in situ phenotypic and karyotypic characterization, which includes an examination of both protein expression and aneuploid chromosomes, has demonstrated its unique advantage for both effective distinguishing and comprehensive detection of CTCs and CTECs. Methods: A total of 98 subjects were recruited in the current study, including healthy donors and patients with benign disease and early‐stage non‐small‐cell lung cancer (NSCLC). SE‐iFISH was performed to quantitatively analyze diverse subtypes of aneuploid CD31+ CTECs and CD31− CTCs classified upon the ploidy of chromosome 8 and tumor marker expression in the specimens collected from the recruited subjects. Results: CD31− CTCs primarily consist of triploid CTCs with a small cell size (≤5 µm) and large hyperploid CTCs (≥ pentaploid), whereas CD31+ CTECs are mainly comprised of large hyperploid cells. Enumeration of the total numbers of both CTCs and CTECs might help identify malignant nodules with a high sensitivity, whereas quantification of tetraploid CTCs and CTECs specifically exhibited a high specificity for the identification of malignant nodules. Conclusions: Combined detection of the specific subtypes of aneuploid CD31+ CTECs and CD31− CTCs may help to effectively identify malignant nodules with a higher sensitivity and specificity in early stage NSCLC patients. [ABSTRACT FROM AUTHOR]

The application of sludge biochar (SC) materials as efficient catalysts for organic pollutants mineralization via advanced oxidation process meets the good strategy of "make waste profitable". The catalytic oxidations of methyl orange (MO) and pyrene by oxalic acid modified sludge biochar (SC-OA) with and without H 2 O 2 were carried out. The analysis of Fourier transform infrared (FT-IR), X-ray photoelectron spectroscopy (XPS), electronic paramagnetic resonance spectrometer (EPR) and free radical quenching experiment were performed and the definite relationships between persistent free radicals (PFRs) type and specific reactive oxygen species (ROS) were made clear. It is suggested for the first time that carbon-centered type PFRs in SC-OA without H 2 O 2 could form O 2 •- and •OH from COOH groups, while oxygen-centered type PFRs induced H 2 O 2 to produce •OH. The degradation intermediates of MO and pyrene were identified and the transformation pathways were proposed. SC-OA, possessing good sustainable utilization and clean catalytic property, is expected to be popularized and applied in the mineralization of organic pollutants, especially in the in-situ remediation of contaminated soil where is no continuous supply of H 2 O 2. It is known that the g-factors for oxygen-centered radicals are larger than 2.0040, while g-factors less than 2.0030 correspond to carbon-centered radicals. As shown in Fig. 4a, the g-factors for SC, SC-OA, and SC-OA deg were 2.1326, 1.9914, and 2.1330, indicating that carbon-centered PFRs played a key role in the catalytic degradation of MO. From the above results, we can speculate that SC has the intrinsic oxygen-centered type PFRs, and OA monoanion on the SC-OA can convert to carbon-centered PFRs (SC–OH/HOOC•) under the activation of the oxygen-centered PFRs followed by radical decarboxylation to give CO 2 and formate. Then, SC-OH/HOOC• stimulates the generation of ROS. [Display omitted] • The relationships of PFRs type and ROS generation was made clear. • Carbon-centered type PFRs in SC-OA prefer to form O 2.•- and •OH without H 2 O 2. • Oxygen-centered type PFRs in SC contribute to induce H 2 O 2 to produce.•OH. • SC-OA possess good sustainable utilization and clean catalytic property. [ABSTRACT FROM AUTHOR]

Cancer stem cells (CSCs), which comprise a small proportion of total cancer cells, have special capacities for self-renewal, differentiation and tumor formation. Currently, CSCs are regarded as the major cause of the failure in anticancer therapy, such as chemoresistance and/or radioresistance, tumor recurrence and metastasis. Autophagy, a process of cellular self-digestion and response to stress, has a role in tumor formation and progression, and it may play a dual role in CSCs-related resistance to anticancer therapy. Most researchers believe that autophagy contributes to stemness maintenance of CSCs and is responsible for the failure of anticancer therapy. Unexpectedly, several studies have also suggested that loss of stemness in CSCs could be mediated by autophagy. Here, we review the recent advances in CSCs and autophagy, especially analyze the complex relationship between them, and hope to apply this new knowledge to the strategies for anticancer therapy. [ABSTRACT FROM AUTHOR]

The effect of hot working parameters on the microstructure and texture evolution of the hot-deformed Zr-45Ti-5Al-3V alloy was studied by the electron backscatter diffraction (EBSD) technique. It was found that a high density of dislocations were generated when the alloy was deformed at 700 °C/0.001 s−1 and 800 °C/1 s−1. With the increment in hot-deformation temperature and the decrease in strain rate, the dislocation density decreased due to the increase in dynamic recrystallization (DRX) degree. The discontinuous dynamic recrystallization (DDRX) and continuous dynamic recrystallization (CDRX) mechanisms co-existed during the hot working of the Zr-45Ti-5Al-3V alloy at a true strain of 0.7. The texture evolution of the alloy during hot working was characterized and the texture component mainly consisted of {001}<100>, {011}<100>, {110}<112>, and {112}<110> textures. The volume fractions of {001}<100> and {011}<100> textures obviously rose with the reduction in strain rate, whereas those of {110}<112> and {112}<110> textures gradually decreased. At a given strain rate, an increase trend in the volume fraction of the {001}<100> texture was observed with rising hot-deformation temperature, while the volume fraction of the {011}<100> texture first increased and then decreased. An opposite trend was visible in the {112}<110> and {110}<112> texture compared with {011}<100> textures. [ABSTRACT FROM AUTHOR]

[Display omitted] • SC-500 exhibited lower triclosan adsorption due to the electrostatic repulsion. • SC-500 exhibited higher triclosan degradation due to higher Fe2+ content to produce more ∙OH. • Aromaticity of dissolved organic matter is positively correlated with the degradation of TCS. When sludge biochar (SC) prepared from sewage sludge was used to remove triclosan (TCS) from both aqueous solution and soil, the adsorption capacity increased with increasing pyrolysis temperature, while the degradation rate showed a different pattern, namely, SC-500 > SC-800 > SC-600 > SC-700. TCS degradation rate achieved nearly 100 % at 2 h with SC-500 used as a Fenton catalyst. Aromaticity of dissolved organic matter (DOM) was positively correlated with the degradation of TCS and adding homologous DOM solution shortened the equilibrium time of TCS degradation, while removing DOM from SC prolonged, compared to the original SC. The results of X-ray photoelectron spectroscopy (XPS) and electron paramagnetic resonance spectrometer (EPR) characterization indicated that SC-500 performed a stronger catalytic degradation ability due to the relatively high Fe2+ content to produce more ∙OH and SC-derived DOM act as an electron shuttle to promote other reactive oxidative species generation through two free radical pathways in the activation of H 2 O 2 by Fe2+, which in turn promoted the degradation of TCS. [ABSTRACT FROM AUTHOR]

• Protective autophagy induced by DDP decreases chemosensitivity in GC cells. • MGMT inhibited ATG4B to repress autophagy. • Inhibition of autophagy enhanced the chemosensitivity to DDP in GC xenograft model. • High MGMT expression and low ATG4B expression are significantly correlated with survival of GC. Cisplatin (DDP) is the first-line drug for the treatment of gastric cancer (GC). However, DDP resistance is common. Autophagy, which is closely related to chemoresistance, is a process of resolving and recycling proteins and damaged cellular organs. Additionally, O-6-methylguanine-DNA methyltransferase (MGMT) is responsible for alkylating drug resistance. However, the relationship between autophagy and MGMT in response to DDP in GC is still unknown. In the present study, we determined that autophagy induced by DDP decreases chemosensitivity in GC cell lines. DDP may have induced autophagy in GC by inhibiting MGMT to increase autophagy-related gene (ATG) 4B. Inhibition of MGMT-mediated ATG4B suppression resulted in autophagy induction and DDP resistance. In vivo , combined DDP and autophagy inhibitor chloroquine (CQ) enhanced the anti-tumor effect of DDP; additionally, the negative correlation of MGMT and ATG4B was confirmed. High expression of MGMT and low expression of ATG4B were significantly correlated with favorable five-year survival rate (P < 0.05) in 66 clinicopathologically characterized GC cases. Our study demonstrate that DDP inhibits MGMT-mediated autophagy suppression to decrease chemosensitivity in GC, which provides a novel therapeutic strategy to promote DDP chemosensitivity in GC. [ABSTRACT FROM AUTHOR]

Low temperature, nitric oxide (NO)/nitrous oxide (N[sub 2]O) aided, sub-35 Å Si[sub 0.85]Ge[sub 0.15] oxynitrides have been grown at 550 and 650 °C, while the oxynitridation feed gases have been preheated to 900 and 1000 °C, respectively, before entering the reaction zone. X-ray photoelectron spectroscopy and secondary ion mass spectroscopy (SIMS) data suggest that NO-assisted oxynitridation incorporates more nitrogen than the N[sub 2]O-assisted one, while there is minimal Ge segregation towards the dielectric/substrate interface in both oxynitridation processes. Moreover, SIMS results suggest that nitrogen is distributed throughout the film in contrast to high temperature Si oxynitridation, where nitrogen incorporation takes place near the dielectric/substrate interface. Z-contrast imaging with scanning transmission electron microscopy shows that the oxynitride grown in NO at 650 °C has a sharp interface with the bulk Si[sub 0.85]Ge[sub 0.15], while the roughness of the dielectric/Si[sub 0.85]Ge[sub 0.15] substrate interface is less than 2 Å. These results are discussed in the context of an overall mechanism of SiGe oxynitridation. [ABSTRACT FROM AUTHOR]

A novel eco-friendly material (CS-U@PS) for persulfate slow-release to effectively degrade organic pollutants (methyl orange and pyrene) was synthesized using chitosan and urea as the encapsulated framework materials via an emulsion cross-linking method for the first time. The obtained CS-U@PS exhibits spherical shapes with a uniform size of approximately 2–3 µm according to the particle-size distribution and SEM image results. The slow-release mechanism was proposed through a kinetics model study and the Ritger–Peppas model fit well (r2 = 0.9699) to indicate that the slow-release process is non-Fickian diffusion. The influences of urea and PS dosages and oxidative conditions on methyl orange degradation were studied, and all the results suggested that urea played an important role in PS slow-release and can also catalyze the activation of PS by iron to further produce radicals and improve the removal efficiency of pollutants. A pyrene removal rate of 90.53% was achieved in aqueous solutions and an above 80% removal rate was obtained in weakly acidic or neutral soil environments by CS-U@PS activated by Fe2+ with citric acid as the chelating agent. Therefore, the fabricated slow-release oxidation materials exhibit application potential for the remediation of organic polluted groundwater and soil. The degradation of MO can be inferred to possibly follow two main pathways as a result of preferential attack of ROS (mainly OH• and SO 4 ⋅ −) on MO molecules. The peak at m/z = 304 is the parent molecule of MO after losing Na+ due to dissolution in aqueous solution. Therefore, from this peak, it was confirmed that MO was not fully degraded. Initially, OH• oxidized the parent MO molecule to form the oxidation product of MO (m/z = 337.2). The other degradation pathway is that the N C bond of the dimethylamino group is broken down, leading to the replacement of the methyl group with protons to form new products corresponding to MO losing two methyl groups (m/z = 277). Then, the peaks found at m/z 139 and 173 proved the symmetric cleavage of the azo bond. The oxidative cleavage of the C and S bonds of the MO dye by free radicals generated the intermediate (m/z = 197.1). The peak at m/z 96.9 is ascribed to the formation of 2-cyclohexen-1-amine, and the peak at m/z 110 can be produced by hydroxylation. Further oxidation of hydroquinone resulted in the destruction (m/z = 171.9) and opening of aromatic rings and the formation of short linear aliphatic carboxylic acids (m/z = 116). Finally, aliphatic carboxylic acids can be directly converted into CO 2 and H 2 O. [Display omitted] • CS-U@PS is a novel chitosan-urea encapsulated material for persulfate slow-release. • The slow-release process is non-Fickian diffusion. • Urea was an excellent and vital sustained release component. • A moderate amount of urea can catalyze the Fe2 + activation of PS to further produce free radicals. • A pyrene removal rate of 90.53% and 80% were achieved in aqueous solutions and soil, respectively. [ABSTRACT FROM AUTHOR]

Abstract: Nitric oxide (NO) aided Si0.85Ge0.15 wet-oxynitridation has been performed at 400–700 °C, while the wet-NO feed gas was preheated to higher temperatures before entering the reaction zone. X-ray photoelectron spectroscopy (XPS) and secondary ion mass spectroscopy data suggests that both nitrogen and oxygen incorporation increases within the dielectric bulk with increasing wet-oxynitridation temperature, while there is no apparent germanium segregation towards the dielectric/substrate interface at all temperatures studied. Moreover, angle-resolved XPS analysis suggests that wet-oxynitridation at temperatures higher than 600 °C volatilizes some germanium oxide from the surface region, while silicon monoxide is outgassed from the dielectric bulk and accumulates near the surface. Nitrogen incorporation is found to hinder germanium segregation. Z-contrast imaging with scanning transmission electron microscopy shows that oxynitrides grown in wet-NO have sharp interfaces with bulk Si0.85Ge0.15, while the roughness of the dielectric/Si0.85Ge0.15 substrate interface is less than 2 Å. These results are discussed in the context of an overall mechanism of SiGe wet-oxynitridation. [Copyright &y& Elsevier]

Dysregulated expression of S100A7 is found in several cancers and plays an important role in tumor progression; however, its carcinogenic role in esophageal squamous carcinoma (ESCC) is still poorly understood. Here, we identified that the levels of S100A7 were remarkably upregulated in 341 tumor tissues (P <.001) and 274 serum samples (P <.001) of ESCC patients compared with normal control. It was an independent prognostic factor (P =.026). Furthermore, a new diagnostic model for ESCC based on serum S100A7, SCC, and crfra21‐1 was established with area under curve (AUC) up to 0.863 (95% CI: 0.802‐0.925). Mechanically, we found upregulated S100A7 could promote cell migration and proliferation through intracellular binding to JAB1 and paracrine interaction with RAGE receptors and then activates the downstream signaling pathways. In addition, exocrine S100A7 could promote M2 macrophage infiltration and polarization by up‐regulating M2 macrophage associated proteins, and tumor angiogenesis by enhancing the activation of p‐ErK and p‐FAK pathways. Further animal experiments confirmed the role of S100A7 in promoting M2 macrophage infiltration and angiogenesis in ESCC. In conclusion, these findings highlighted the potential diagnostic and prognostic value of S100A7 in patients with ESCC. Meanwhile, our results reveal that S100A7 promotes tumor progression by activating oncogenic pathways and remodeling tumor microenvironment, which paving the way for the progress of S100A7 as a therapeutic target for cancer treatment. [ABSTRACT FROM AUTHOR]

Along with the gradually increasing incidence, nonalcoholic fatty liver disease (NAFLD) has already been influencing the health of more and more people in the world. Corydalis saxicola Bunting (CSB), a valuable folk medicine, is the dried whole grass of a perennial herb, Yanhuanglian (Papaveraceae), which has significant effects on various hepatitis, liver fibrosis, cirrhosis and other liver diseases. Corydalis saxicola Bunting total alkaloids (CSBTA), a mixture of alkaloids extracted from CSB, exhibit widely-accepted hepatoprotective effects. This study aimed to explore the therapeutic potential of CSBTA on NAFLD and the underlying mechanism. A mice model was established by high fat and high cholesterol diet (HFHCD) to study the benefits of CSBTA on the progression of NAFLD. The efficacy of CSBTA on NAFLD was revealed systematically via RNA-sequencing analysis. Further efficacy and molecular mechanism study were explored in mouse primary hepatocytes and HepG2 cells stimulated with high energy with or without pharmacological inhibition or gene silencing. CSBTA effectively improved the major hallmarks of NAFLD including liver lipid accumulation, liver injury, inflammation and fibrosis in HFHCD-fed mice. RNA sequencing and targeted qPCR analysis jointly evidenced CSBTA significantly suppressed the expression of Srebf1 , Acc1 and Fasn which are the genes responsible for fatty acid biosynthesis. Moreover, stable isotope tracer test denoted CSBTA reduced lipid accumulation via interrupting fatty acid biosynthesis in hepatocytes or the liver. Mechanistically, CSBTA could impede SREBP1 maturation via AMPK activation, thereby reducing DNL-derived lipid accumulation in hepatocytes. CSBTA protected against hepatic steatosis and other hallmarks of NAFLD induced by HFHCD via suppressing DNL, through modulating the AMPK-SREBP1 axis. CSBTA may therefore have a therapeutic potential for NAFLD treatment. [Display omitted] • CSBTA ameliorated high energy diet induced NAFLD in mice. • CSBTA reduced DNL-derived lipid accumulation via regulating hepatic AMPK-SREBP1 pathway. • CSBTA may be a therapeutic candidate for the treatment of NAFLD. [ABSTRACT FROM AUTHOR]

Purpose: Lung adenocarcinoma (LUAD) is the predominant subtype of lung cancer, with increasing evidence showing clinical benefits of immunotherapy. However, a lack of integrated profiles of complex LUAD immune microenvironments hampers the application of immunotherapy, resulting in limited eligible patient populations as well as drug resistance problems. Here, we aimed to systematically profile the immune signatures of LUADs and to assess the role of the immune microenvironment in patient outcome. Methods: We systematically profiled the immune signatures of LUADs deposited in the TCGA and GEO databases using a total of 730 immune-related genes. Differential expression analysis was used to identify dysregulated genes. Univariate Cox analysis followed by robust likelihood-based survival analysis and multivariate Cox analysis were applied to construct an immune-related prognostic model. Results: We found that differentially expressed immune genes were mainly enriched in immune cell proliferation, migration, activation and the NF-κB and TNF signaling pathways. The 10-immune gene predictive model that we constructed could differentiate LUAD patients with different overall survival times in several datasets, with areas under the curve (AUCs) of 0.67, 0.69, 0.72 and 0.74. LUAD patients with high- or low-risk scores exhibited distinct immune cell compositions, which may explain the prognostic significance of our model. Conclusions: Our results add to the current knowledge of immune processes in LUADs and underscore the critical role of the immune microenvironment in LUAD patient outcome. [ABSTRACT FROM AUTHOR]

Correlation between fecal calprotectin (FC) and endoscopic activity assessed by Ulcerative Colitis Endoscopic Index of Severity (UCEIS) in acute severe colitis (ASC) patients was explored to evaluate the predictive value of FC in clinical outcomes. Seventy-one ASC patients were retrospectively evaluated. FC level within 3 days of colonoscopy was measured with ELISA. Demographic and clinical data, laboratory parameters, and medical therapy were documented, and the endoscopic severity of disease was rated by UCEIS. The end points were the rate of failed corticosteroid therapy, colectomy, and mortality. There was significant correlation between UCEIS and FC level (r=0.729, P<0.001), which was superior to CRP, ESR, and hemoglobin. FC level between endoscopic mild activity (UCEIS, 3-4) and endoscopic moderate activity (UCEIS, 5-6), and endoscopic moderate activity (UCEIS, 5-6) and endoscopic severe activity (UCEIS, 7-8) were significantly different. FC levels were associated with different outcomes (failed corticosteroid therapy or surgery); when FC >1672 µg/g, sensitivity and specificity were 80.2 and 66.7%, respectively, in prediction for colectomy using receiver operating characteristics analysis. The results indicated that FC, as a non-invasive indicator, correlates positively with the UCEIS. Baseline FC level predicts clinical outcomes in ASC patients, which make a timely treatment strategy conversion possible after accurately forecasting the likelihood of failure of intravenous steroid therapy. [ABSTRACT FROM AUTHOR]

Type 2 diabetes mellitus (T2DM), characterized by insulin resistance and unclear pathogenesis, is a serious menace to human health. Bile acids are the end products of cholesterol catabolism and play an important role in maintaining cholesterol homeostasis. Furthermore, increasing studies suggest that bile acids may regulate glucose tolerance, insulin sensitivity, and energy metabolism, suggesting that bile acids may represent a potential therapeutic target for T2DM. This study summarizes the metabolism of bile acids and, more importantly, changes in their concentrations, constitution, and receptors in diabetes. Furthermore, we provide an overview of the mechanisms underlying the role of bile acids in glucose and lipid metabolism, as well as the occurrence and development of T2DM. Bile acid-targeted therapy may represent a valid approach for T2DM treatment. [ABSTRACT FROM AUTHOR]

The gut microbiota forms a symbiotic relationship with the host and benefits the body in many critical aspects of life. However, immune system defects, alterations in the gut microbiota and environmental changes can destroy this symbiotic relationship and may lead to diseases, including cancer. Due to the anatomic and functional connection of the gut and liver, increasing studies show the important role of the gut microbiota in the carcinogenesis of hepatocellular carcinoma (HCC). In this manuscript, we review the available evidence and analyze some potential mechanisms of the gut microbiota, including bacterial dysbiosis, lipopolysaccharide (LPS), and genotoxins, in the progression and promotion of HCC. Furthermore, we discuss the possible therapeutic applications of probiotics, chemotherapy modulation, immunotherapy, targeted drugs and fecal microbiota transplantation (FMT) in targeting the gut microbiota. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitors against PD-1/PD-L1 yield improved survival rates of KRAS-mutant NSCLC patients, who conferred a poor prognosis without effective targeted therapy until now. Yet, the underlying association between KRAS mutations and immune responses remains unclear. We performed an integrated analysis of the data from publicly available repositories and from clinical center cohorts to explore the association between KRAS mutation status and tumor immunity-associated features, including PD-L1 expression, CD8+ tumor-infiltrating lymphocytes (TILs) and tumor mutational burden (TMB). Our results revealed that KRAS mutations are correlated with an inflammatory tumor microenvironment and tumor immunogenicity, resulting in superior patient response to PD-1/PD-L1 inhibitors. Meanwhile, three-pool analysis further confirmed that KRAS-mutant NSCLC patients show remarkable clinical benefit from anti-PD-1/PD-L1 immunotherapy. In addition, a KRAS-mutant lung adenocarcinoma mouse model was established to estimate the relative efficacy of anti-PD-L1 monoclonal antibody monotherapy or combination treatment with docetaxel versus docetaxel alone. Most surprisingly, we found that PD-L1 blockade combined with docetaxel did not promote an anti-tumor response. These findings uncover that PD-1/PD-L1 blockade monotherapy may be the optimal therapeutic schedule in NSCLC patients harboring KRAS mutations. [ABSTRACT FROM AUTHOR]

Background: Lung cancer is the leading cause of cancer-related death worldwide, and the timely and serial assessment of low-dose computed tomography (LDCT) in high-risk populations remains a challenge. Furthermore, testing a single biomarker for the diagnosis of lung cancers is of relatively low effectiveness. Thus, a stronger diagnostic combination of blood biomarkers is needed to improve the diagnosis of non-small cell lung cancer (NSCLC).Methods: The blood levels of individual biomarkers [IDH1, DNA methylation of short stature homeobox 2 gene (SHOX2), and prostaglandin E receptor 4 gene (PTGER4)] were measured and statistically analyzed in samples from healthy controls and patients with lung cancer. In total, 221 candidates were enrolled and randomly assigned into two groups for the training and validation of a diagnostic panel. Additionally, a subgroup analysis was performed in the whole cohort.Results: A newly combined 3-marker diagnostic model for lung cancers was established and validated with area under the receiver operating characteristic (ROC) curve (AUC) values ranging from 0.835 to 0.905 in independent groups showing significantly stronger diagnostic value compared with a single tested biomarker. The sensitivity of the diagnostic model was as high as 86.1% and 80.0% in the training and validation sets, respectively. Although no apparent differences were found between the 3-marker and 2-marker models, the high clinical T-stage and histological type specificity of IDH1 and two other methylated DNA biomarkers were demonstrated in the subgroup analysis.Conclusions: The combination of single biomarkers with high stage-specificity and histological type specificity (SHOX2 and PTGER4 DNA methylation and IDH1) showed better diagnostic performance in the detection of lung cancers compared with single marker assessment. A greater clinical utility of the panel may be developed by adding demographic/epidemiologic characteristics. [ABSTRACT FROM AUTHOR]

Objectives: Although low-dose computed tomography has been confirmed to have meaningful diagnostic utility, lung cancer is still the leading cause of cancer-related deaths for both genders worldwide. Thus, a novel panel with a stronger diagnostic performance for lung cancer is needed. This study aimed to investigate the efficacy of a new panel in lung cancer diagnosis. Materials and Methods: The serum levels of carcinoembryonic antigen (CEA), cancer antigen 125 (CA125) and seven autoantibodies were measured and statistically analyzed in samples from healthy controls and patients with lung cancer. The 316 candidates enrolled in this study were randomly assigned into two groups for the training and validation of a diagnostic panel. Results: An optimal panel with four biomarkers (CEA, CA125, Annexin A1-Ab, and Alpha enolase-Ab) was established, with an area under the receiver operator characteristic (ROC) curve (AUC) of 0.897, a sensitivity of 86.5%, a specificity of 82.3%, a positive predictive value (PPV) of 88.3%, a negative predictive value (NPV) of 79.7%, and a diagnostic accuracy of 84.8% for the training group. The panel was validated, with an AUC of 0.856 and a sensitivity of 87.5% for the validation group. Furthermore, the new panel performed significantly better in lung cancer screening than did CEA and CA125 in all of the cohorts (p<.05). Conclusion: The diagnostic performance of CEA and CA125 was significantly enhanced through their combination with two autoantibodies (Annexin A1-Ab, and Alpha enolase-Ab). Optimization of the measured autoantibodies is critical for generating a panel to detect lung cancer in patients. [ABSTRACT FROM AUTHOR]

Interferons (IFNs) play crucial roles in the development and treatment of cancer. Long non-coding RNAs (lncRNAs) are emerging molecules involved in cancer progression. Here, we identified and characterized an IFN-inducible nuclear lncRNA IRF1-AS (Interferon Regulatory Factor 1 Antisense RNA) which was positively correlated with IRF1 expression. IFNs upregulate IRF1-AS via the JAK-STAT pathway. Knockdown and overexpression of IRF1-AS revealed that IRF1-AS inhibits oesophageal squamous cell carcinoma (ESCC) proliferation and promotes apoptosis in vitro and in vivo. Mechanistically, IRF1-AS activates IRF1 (Interferon Regulatory Factor 1) transcription through interacting with ILF3 (Interleukin Enhancer Binding Factor 3) and DHX9 (DExH-Box Helicase 9). In turn, IRF1 binds to the IRF1-AS promoter directly and activates IRF1-AS transcription. Global analysis of IRF1-AS-regulated genes indicated that IRF1-AS activates the IFN response in vitro and in vivo. IRF1 knockdown in IRF1-AS-overexpressing cells abolished the antiproliferative effect and activation of the IFN response. Furthermore, IRF1-AS was downregulated in ESCC tissues, and low expression correlated with poor prognosis. In conclusion, the interferon-inducible lncRNA IRF1-AS represses esophageal squamous cell carcinoma progression by promoting interferon response through a positive regulatory loop with IRF1. [ABSTRACT FROM AUTHOR]

Purpose: Hub genes are good molecular candidates for targeted cancer therapy. As yet, however, there is little information on the clinical implications and functional characteristics of hub genes in the development of non-small cell lung cancer (NSCLC). In this study, we set out to analyze the role of hub genes in NSCLC. Methods: We performed weighted gene co-expression network analysis (WGCNA) to analyze gene networks during NSCLC development using transcriptomic data from normal, pre-cancer and cancer tissues. Both in vitro and in vivo expression knockdown assays were used to evaluate the biological function of candidate hub gene CDC45 (cell division cycle 45) in NSCLC. Results: We identified 14 gene networks associated with NSCLC development, in which two modules (turquoise and green) correlated with tumorigenesis most positively and negatively, respectively. Gene enrichment analysis showed that the turquoise module was associated with cell cycle/mitosis, and that the green module was associated with development/morphogenesis. We found that the expression levels of the hub genes CDC45, CDCA5, GINS2, RAD51 and TROAP in the turquoise module increased gradually during tumorigenesis, whereas those of MAGI2-AS3 and RBMS3 in the green module decreased during tumorigenesis. Functionally, we found that expression knockdown of CDC45 inhibited NSCLC cell proliferation both in vitro and in vivo, and arrested the cells in the G2/M phase of the cell cycle, supporting an oncogenic role of CDC45. Conclusion: Through gene co-expression network analysis and subsequent functional analyses we identified hub gene CDC45 as a putative novel therapeutic target in NSCLC. [ABSTRACT FROM AUTHOR]

Traditional Chinese medicine believes that "blood fever" is an important cause of psoriasis. Fufang Shengdi mixture (FFSD), based on the Hongban Decoction, is composed of Rehmannia glutinosa (Gaertn.) DC., Raw gypsum (Chinese: Sheng Shi Gao), and Lonicera japonica Thunb (Caprifoliaceae). FFSD has effects on nourishing Yin, clearing heat, connecting collaterals, and cooling blood. In modern medical explanation, FFSD has the effects of anti-inflammatory and immunosuppression. Our study proved that FFSD can suppress immunity and ameliorate the symptoms of imiquimod-induced psoriasis in mice. This study evaluated the efficacy and possible mechanism of FFSD in psoriasis mice. First, the main components of FFSD were analyzed using high-performance liquid chromatography-tandem high-resolution mass spectrometry (HPLC-HRMS). An imiquimod (IMQ)-induced psoriasis mouse model was used to evaluate the efficacy of FFSD orally. Psoriasis area and severity index (PASI) scores were recorded throughout the course of the mice to reflect the severity of psoriasis. Hematoxylin-eosin staining was used to observe the pathological changes in skin lesions. Enzyme-linked immunosorbent assay (ELISA) was performed to test the level of IFN-γ and TNF-α in plasma. To further investigate the immunopharmacological effect of FFSD, we used chicken ovalbumin (OVA) to induce immunoreaction in mice. ELISA was used to detect the levels of anti-OVA antibody, IFN-γ and TNF-α in mice. Flow cytometry was performed to quantify the ratio of cell types in peripheral blood mononuclear cells (PBMCs) to evaluate the effect of FFSD on immunosuppression. Proteomics and bioinformatics analyzes were performed to find the regulation pathway of the immunosuppressive effect of FFSD. Finally, quantitative PCR (qPCR) and immunohistochemistry were used to measure the upregulation of Annexin-A proteins (ANXAs) in the skin lesion tissue of IMQ-induced mouse. On the basis of knowing the composition of FFSD, we first proved the efficacy of FFSD in alleviating IMQ-induced psoriasis in mice. Second, we further clarified the pharmacological effect of FFSD on immunosuppression via OVA-induced mice. Subsequently, it was found that the significant up-regulation of ANXAs was caused by FFSD through proteomics analysis, and the finding was proved in the IMQ-induced psoriasis mouse model. This study elucidates the immunosuppressive pharmacological effect of FFSD on improving psoriasis through up-regulating ANXAs. [Display omitted] [ABSTRACT FROM AUTHOR]

Alternative splicing (AS) and the regulation of AS by splicing factors play critical roles in cancer. Plant homeodomain (PHD)–finger domain protein PHF5A, a critical splicing factor involved in AS, has been demonstrated to play an oncogenic role in glioblastoma multiforme and breast cancer, but its biological function in lung cancer remains unclear. In the present study, we systematically analyzed the biological function and clinical relevance of PHF5A in non–small cell lung cancer (NSCLC). We found that PHF5A was significantly upregulated in NSCLC tumors compared with normal tissues in both TCGA data set and tissue microarrays. Upregulation of PHF5A was negatively correlated to the overall survival (OS) of lung adenocarcinoma (LUAD) patients. Loss‐of‐function and gain‐of‐function experiments confirmed that PHF5A functioned as an oncoprotein by promoting LUAD cell proliferation, migration and invasion, inducing G0/G1 cell cycle progression and inhibiting cisplatin–induced apoptosis. RNA–seq analysis identified many essential genes whose AS was dysregulated by PHF5A, including cell cycle–associated genes such as SKP2, CHEK2, ATR and apoptosis–associated genes such as API5 and BCL2L13. Additionally, pladienolide, a small molecular inhibitor of PHF5A, inhibited LUAD cell proliferation in a dose–dependent manner and induced AS changes similar to PHF5A knockdown. In conclusion, we validated that PHF5A played an oncogenic role via AS in LUAD and suggested that PHF5A might serve as a potential drug target with a promising anticancer therapeutic effect.We demonstrated that PHF5A acted as a key regulator in the AS of many essential genes to promote the development and progression of LUAD. PHF5A might serve as a potential drug target with a promising anticancer therapeutic effect in LUAD patients. [ABSTRACT FROM AUTHOR]

Alternative splicing (AS), a major mechanism for the enhancement of transcriptome and proteome diversity, has been widely demonstrated to be involved in the full spectrum of oncogenic processes. High-throughput sequencing technology and the rapid accumulation of clinical data sets have provided an opportunity to systemically analyze the association between messenger RNA AS variants and patient clinical outcomes. Here, we compared differentially spliced AS transcripts between esophageal carcinoma (ESCA) and non-tumor tissues, profiled genome-wide survival-associated AS events in 87 patients with esophageal adenocarcinoma (EAC) and 79 patients with esophageal squamous cell carcinoma (ESCC) using The Cancer Genome Atlas (TCGA) RNA-seq data set, and constructed predictive models as well as splicing regulation networks by integrated bioinformatic analysis. A total of 2326 AS events in 1738 genes and 1812 AS events in 1360 genes were determined to be significantly associated with overall survival (OS) of patients in the EAC and ESCC cohorts, respectively, including some essential participants in the oncogenic process. The predictive model of each splice type performed reasonably well in distinguishing good and poor outcomes of patients with esophageal cancer, and values for the area under curve reached 0.942 and 0.815 in the EAC exon skip predictive model and the ESCC alternate acceptor site predictive model, respectively. The splicing regulation networks revealed an interesting correlation between survival-associated splicing factors and prognostic AS genes. In summary, we created prognostic models for patients with esophageal cancer based on AS signatures and constructed novel splicing correlation networks. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lncRNAs) play critical roles in multiple cellular processes in non-small cell lung cancer (NSCLC); however, the involvement of lncRNAs in the transforming growth factor-beta (TGFβ) signaling pathway, the critical tumor cell epithelial-mesenchymal transition (EMT) and metastasis pathway, remains poorly understood. To address this issue, we compared the lncRNAs expression patterns of NSCLC cells treated with and without TGFβ1 treatment. We observed that one of the most prominent hits, TGFβ-induced lncRNA (TBILA), promoted NSCLC progression and was upregulated in tumor tissues. Upregulated TBILA promotes human germinal center-associated lymphoma (HGAL) expression by binding to the Smad transcription factor complex, thereby enhancing RhoA activation. In addition, TBILA induces the S100A7-c-Jun activation domain-binding protein 1 (JAB1) pathway by binding to nuclear S100A7 and enhances pro-survival pathways in NSCLC. These findings have provided us with a new perspective regarding the regulation of the TGFβ signaling pathway in NSCLC and suggest that the lncRNA TBILA can serve as a target for anticancer therapies. [ABSTRACT FROM AUTHOR]

Abstract: The long non‐coding RNA GAS5 has been reported as a tumor suppressor in many cancers. However, its functions and mechanisms remain largely unknown in esophageal squamous cell carcinoma (ESCC). In this study, we found that GAS5 was over‐expressed in ESCC tissue compared with that in normal esophageal tissue in a public database. Functional studies showed that GAS5 could inhibit ESCC cell proliferation, migration and invasion in vitro. Further analysis revealed that GAS5 was regulated by interferon (IFN) responses via the JAK‐STAT pathway. Moreover, as an IFN‐stimulated gene (ISG), GAS5 was a positive regulator of IFN responses. The feedback loop between GAS5 and the IFN signaling pathway plays an important antitumor role in ESCC, thus providing novel potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Abstract: The transforming growth factor β (TGF-β) signaling pathway plays anti- and pro-tumoral roles in the vast majority of cancers, and long noncoding RNAs have been reported to play key roles in the highly contextual response process. However, the roles of long noncoding RNAs (lncRNAs) in TGF-β signaling in esophageal squamous cell carcinoma (ESCC) remain unknown. In this study, we performed RNA-seq to compare lncRNAs expression levels between TGF-β1-treated and untreated ESCC cells and observed that NF-kappaB-interacting lncRNA (NKILA) was remarkably upregulated by the classical TGF-β signaling pathway. RNA profiling of 39 pairs ESCC tumor and adjacent nontumor samples using RT-qPCR demonstrated that NKILA is significantly downregulated in ESCC tumor tissues, and NKILA expression levels were significantly decreased in advanced tumor tissues (III and IV) compared to early stages (I and II) (p < 0.01). Gain- and loss-of-function assays showed that NKILA inhibited ESCC cell metastasis in vitro and in vivo, and mechanism studies showed that NKILA repressed MMP14 expression by inhibiting IκBα phosphorylation and NF-κB activation. Collectively, these findings suggest that the TGF-β-induced lncRNA NKILA has potential as an antimetastasis therapy.Key messages: Long noncoding RNA NKILA could be remarkably upregulated by classical TGF-β signal pathway in ESCC.NKILA was significantly downregulated in esophageal squamous cell carcinoma and negatively correlated with TNM stage.NKILA inhibits ESCC cell metastasis via repressing MMP14 expression by suppressing the phosphorylation of IκBα and NF-κB activation. [ABSTRACT FROM AUTHOR]

• Structural semantic information is introduced in LDCT domain • Structural semantic loss function is designed for network training • Semantic evaluation metric is applied for image quality assessment. Background: To reduce radiation exposure and improve diagnosis in low-dose computed tomography, several deep learning (DL)-based image denoising methods have been proposed to suppress noise and artifacts over the past few years. However, most of them seek an objective data distribution approximating the gold standard and neglect structural semantic preservation. Moreover, the numerical response in CT images presents substantial regional anatomical differences among tissues in terms of X-ray absorbency. Methods: In this paper, we introduce structural semantic information for low-dose CT imaging. First, the regional segmentation prior to low-dose CT can guide the denoising process. Second the structural semantical results can be considered as evaluation metrics on the estimated normal-dose CT images. Then, a semantic feature transform is engaged to combine the semantic and image features on a semantic fusion module. In addition, the structural semantic loss function is introduced to measure the segmentation difference. Results: Experiments are conducted on clinical abdomen data obtained from a clinical hospital, and the semantic labels consist of subcutaneous fat, muscle and visceral fat associated with body physical evaluation. Compared with other DL-based methods, the proposed method achieves better performance on quantitative metrics and better semantic evaluation. Conclusions: The quantitative experimental results demonstrate the promising performance of the proposed methods in noise reduction and structural semantic preservation. While, the proposed method may suffer from several limitations on abnormalities, unknown noise and different manufacturers. In the future, the proposed method will be further explored, and wider applications in PET/CT and PET/MR will be sought. [ABSTRACT FROM AUTHOR]

Recent evidence suggests that neuropsychiatric stabilizers have a place in resolving gastrointestinal disorders. Lithium carbonate (LC) is one of the most commonly used drugs for bipolar disorder clinically. Here, we estimate the therapeutic function of LC against colitis and investigate the mechanism of intestinal flora and metabolism modulation. A colitis model was constructed by continuously administering 2.5% dextran sodium sulfate (DSS) solution daily for 7 days. Analysis of gut microbiota was carried out by 16S rRNA gene high-throughput sequencing. Spectrum antibiotic cocktail (ABX) and faecal microbiota transplantation (FMT) were employed to evaluate the protective effect of intestinal flora. Colonic Treg cells and related immune responses were detected by flow cytometry. LC treatment significantly alleviated colon inflammation by regulating gut microbial diversity and altering flora composition. Notably, LC treatment upregulated short-chain fatty acid (SCFA)-producing bacteria, especially Akkermansia muciniphila (A. muciniphila), and transformed metabolite SCFA profiles. LC activated anti-inflammatory Treg cell responses in colonic lamina propria (LP) in a G-protein coupled receptor 43 (GPR43)-dependent mechanism. ABX, FMT and single bacteria gavage experiments were conducted to confirm the above mechanism. As an intestinal microbiome and metabolite modulator, LC alleviates colon inflammation in a GPR43-dependent manner through activating Treg cell responses. Therefore, the therapeutic strategy of the microbiome-metabolite-immune axis, as observed in the A. muciniphila -SCFA-Treg cell axis in our study, might provide a new direction for the treatment of IBD. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: Mycoplasma bovis (M. bovis) is a highly contagious cattle pathogen spreading worldwide and especially in Ningxia Hui Autonomous Region in China. Results: Two types of ST, ST10and ST134, were identified in Ningxia Hui Autonomous Region. Thirty-seven strains belonged to ST10 and 28 strains belonged to ST134. ST134 was a new ST and first found in 2009 and was only widely distributed in Ningxia Hui Autonomous Region at present. The M. bovis ST10 was widely spread in many provinces in China and was widespread in Ningxia Hui Autonomous Region since 2010. It is speculated that the prevalence of M. bovis ST10 in Ningxia Hui Autonomous Region began in 2010. Conclusions: This study is the first report on the genetic diversity of M. bovis from 2009 to 2018 in Ningxia Hui Autonomous Region and provides the epidemiological information. These results may help further our understanding of the evolution of M. bovis and provide information that may be useful for the development of novel vaccines. [ABSTRACT FROM AUTHOR]

Background: Angiogenesis, a basic requirement for tumor cell survival, is considered to be a malignant characteristic of small cell lung cancer (SCLC) and is closely related to the poor outcomes of SCLC patients. miR-141 has been found to play pro- and antiangiogenic roles in different cancers, but its role in SCLC angiogenesis has never been explored. Methods: Total RNA was isolated from plasm exosomes and serum of SCLC patients to examine the expression of miR-141 by qRT-PCR. Cell proliferation, invasion, migration, tube formation assay, aortic ring assay and mouse tumor model were used to investigate the effect of exosomal miR-141 in angiogenesis in vitro and in vivo. Dual-luciferase assay was conducted to explore the target gene of miR-141. Results: Circulating miR-141 was upregulated in samples from 122 SCLC patients compared with those from normal volunteers and that the increase in miR-141 was significantly associated with advanced TNM stages, implying the potential oncogenic role of miR-141 in SCLC malignancy. In vitro, miR-141 that was packaged into SCLC cell-secreted exosomes and delivered to human umbilical vein vascular endothelial cells (HUVECs) via exosomes facilitated HUVEC proliferation, invasion, migration and tube formation and promoted microvessel sprouting from mouse aortic rings. Matrigel plug assays demonstrated that SCLC cell-derived exosomal miR-141 induced neoangiogenesis in vivo. Furthermore, mouse subcutaneous tumor nodules that were developed from miR-141-overexpressing SCLC cells had a higher microvessel density (MVD) and grew faster than those developed from negative control cells. KLF12 was found to be the direct target gene of miR-141 and that the proangiogenic effect of miR-141 on HUVECs was abrogated by KLF12 overexpression. Conclusions: Our results demonstrate the specific function of the exosomal miR-141/KLF12 pathway in SCLC angiogenesis for the first time and provide potential novel targets for antiangiogenic therapies for SCLC patients. [ABSTRACT FROM AUTHOR]

The MiT/TFE transcription factors play a pivotal role in the regulation of autophagy and lysosomal biogenesis. The subcellular localization and activity of MiT/TFE proteins are primarily regulated through phosphorylation. And the phosphorylated protein is retained in the cytoplasm and subsequently translocates to the nucleus upon dephosphorylation, where it stimulates the expression of hundreds of genes, leading to lysosomal biogenesis and autophagy induction. The transcription factor-mediated lysosome-to-nucleus signaling can be directly controlled by several signaling molecules involved in the mTORC1, PKC, and AKT pathways. MiT/TFE family members have attracted much attention owing to their intracellular clearance of pathogenic factors in numerous diseases. Recently, multiple studies have also revealed the MiT/TFE proteins as master regulators of cellular metabolic reprogramming, converging on autophagic and lysosomal function and playing a critical role in cancer, suggesting that novel therapeutic strategies could be based on the modulation of MiT/TFE family member activity. Here, we present an overview of the latest research on MiT/TFE transcriptional factors and their potential mechanisms in cancer. [ABSTRACT FROM AUTHOR]

Understanding interactions between tumor and the host immune system holds great promise to uncover biomarkers for targeted therapies and clinical outcomes. However, systematical analysis of immune signatures in esophageal squamous cell carcinoma (ESCC) remains largely unstudied. In this study, immune signatures containing 708 immune related genes were curated from mRNA microarray data with tumor and paired normal tissues from 119 ESCC patients. Differential expression and survival analysis were performed with validations from Human Protein Atlas and an independent cohort of 110 ESCC patients by immunohistochemistry staining. We identified a total of 186 significantly dysregulated genes in ESCC, including downregulated genes SPINK5, IL1RN and upregulated genes SPP1 and PLAU, which were further confirmed in Human Protein Atlas data. Moreover, nine immune related genes (ABL1, ATF2, ATG5, C6, CD38, HMGB1, ICOSLG, IL12RB2 and PLAU) were significantly associated with patients' overall survival, among which, prognostic model was built including three independent factors ABL1, CD38 and ICOSLG. Validation by immunohistochemistry staining suggested that combination with tumor infiltrated CD4+ and CD8+ T lymphocytes would yield higher performance in distinguishing cases as high or low risk of unfavorable prognosis. In summary, we profiled the immune status in ESCC and established predictive and prognostic factors for ESCC, which could reflect immune disorders within tumor microenvironments and independently distinguish patients with a high risk of reduced survival, providing novel predictive and therapeutic targets for ESCC patients in the future. [ABSTRACT FROM PUBLISHER]