Recapitulating familial hypercholesterolemia in a mouse model by knock-in patient-specific LDLR mutation.

Familial hypercholesterolemia (FH) is one of the most prevalent monogenetic disorders leading to cardiovascular disease (CVD) worldwide. Mutations in Ldlr, encoding a membrane-spanning protein, account for the majority of FH cases. No effective and safe clinical treatments are available for FH. Adenine base editor (ABE)-mediated molecular therapy is a promising therapeutic strategy to treat genetic diseases caused by point mutations, with evidence of successful treatment in mouse disease models. However, due to the differences in the genomes between mice and humans, ABE with specific sgRNA, a key gene correction component, cannot be directly used to treat FH patients. Thus, we generated a knock-in mouse model harboring the partial patient-specific fragment and including the Ldlr W490X mutation. LdlrW490X/W490X mice recapitulated cholesterol metabolic disorder and clinical manifestations of atherosclerosis associated with FH patients, including high plasma low-density lipoprotein cholesterol levels and lipid deposition in aortic vessels. Additionally, we showed that the mutant Ldlr gene could be repaired using ABE with the cellular model. Taken together, these results pave the way for ABE-mediated molecular therapy for FH.

Dynamic Analysis and FPGA Implementation of a New Fractional-Order Hopfield Neural Network System under Electromagnetic Radiation.

Fractional calculus research indicates that, within the field of neural networks, fractional-order systems more accurately simulate the temporal memory effects present in the human brain. Therefore, it is worthwhile to conduct an in-depth investigation into the complex dynamics of fractional-order neural networks compared to integer-order models. In this paper, we propose a magnetically controlled, memristor-based, fractional-order chaotic system under electromagnetic radiation, utilizing the Hopfield neural network (HNN) model with four neurons as the foundation. The proposed system is solved by using the Adomain decomposition method (ADM). Then, through dynamic simulations of the internal parameters of the system, rich dynamic behaviors are found, such as chaos, quasiperiodicity, direction-controllable multi-scroll, and the emergence of analogous symmetric dynamic behaviors in the system as the radiation parameters are altered, with the order remaining constant. Finally, we implement the proposed new fractional-order HNN system on a field-programmable gate array (FPGA). The experimental results show the feasibility of the theoretical analysis.

Kinase D-interacting Substrate of 220 kDa Is Overexpressed in Gastric Cancer and Associated With Local Invasion.

BACKGROUND/AIM: Kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning protein (ARMS), is a transmembrane scaffold protein. Deregulated Kidins220 has been observed in various malignancies including melanoma, glioma, neuroblastoma, prostate cancer, pancreatic cancer, and ovarian cancer. MATERIALS AND METHODS: In the current study, Kidins220 expression was determined at transcript and protein levels. A Kidins220 knockdown cell model was established to identify its role in cellular functions including cell cycle, proliferation, and invasion. Cell signalling was analysed by protein array and the TCGA gastric cancer cohort. RESULTS: Kidins220 transcript levels were significantly increased in gastric tumours, compared with adjacent normal tissues. More advanced tumours (TNMIII and TNMIV) exhibited higher protein levels of Kidins220 compared with early-stage tumours (TNMI and TNMII). Increased expression of Kidins220 in gastric cancer was associated with poorer overall survival. Loss of Kidins220 promoted cell invasion and adhesion of gastric cancer and correlated to epithelial-mesenchymal transition (EMT) and matrix metalloproteinase (MMP) signalling. Knockdown of Kidins220 promoted proliferation of gastric cancer cells with an increased population at the G2/M phase. CONCLUSION: Our study identified increased expression of Kidins220 in gastric cancer, which is associated with disease progression and poor prognosis. However, Kidins220 presented an inhibitory effect on the proliferation, invasion, and adhesion through a regulation of EMT, MMP and cell cycle.

COL10A1 is a potential immunotherapy biomarker associated with immune infiltration and deficient mismatch repair in colon cancer.

Aim: Our study aimed to identify the role of COL10A1 in colon cancer, including interaction with immune infiltrates and somatic mutations. Methods: COL10A1 expression and prognostic value were assessed. Correlations between COL10A1 and various immune parameters were conducted by bioinformatic analysis. Results: Our study demonstrated that COL10A1 is overexpressed in colon cancer and correlates with poor patient survival. The expression level of COL10A1 is significantly associated with mismatch repair deficiency and immune infiltration. High expression of COL10A1 may confer greater sensitivity to anti-PD-1 treatment in colon cancer patients. Conclusion: COL10A1 is a potential diagnostic biomarker associated with deficient mismatch repair and immune infiltration in colon cancer.

Colorectal cancer (CRC) is a deadly disease and we do not have a cure. Immunotherapy is a new method that can help CRC patients to live longer, but it only works for some people. To find out who will get good results with immunotherapy, we looked at a protein named COL10A1. We found more COL10A1 in colon cancer tissues than in healthy tissues. CRC patients with a lot of COL10A1 are more likely to die than those patients with low levels of this protein. COL10A1 can interact with some immune cells and by looking at how much COL10A1 is in different CRC patients, we may be able to choose the right patients to treat with immunotherapy.

Identification and application of species-specific peptide biomarkers from soft-shelled turtles (Pelodiscus sinensis) using post-translational modification detection-based peptidomics analysis.

The soft-shelled turtle is a commercially aquatic species in Asian countries, which serves as an important source of collagen with high nutritional and medicinal value, so it is of great significance to distinguish soft-shelled turtle derived collagen from others or adulterations. In this work, peptidomics analysis based on post-translational modification (PTM) assay was used to discover specific peptide biomarkers of soft-shelled turtle gelatin (STG). In total eight specific sequences and 74 peptides with different PTM types were screened out, and seven peptides with good signal responses and STG specificity were selected and validated as STG-specific peptide biomarkers. These peptide biomarkers could be used for distinguishing STG from other animal gelatins, and applied for ensuring the quality of collagens or gelatins from soft-shelled turtle with authenticity and traceability.

Iron (oxyhydr)oxides are responsible for the stabilization of Cu and Zn in AMD after treatment with limestone.

The formation and transformation of secondary iron (oxyhydr)oxides and their role in the stabilization of copper (Cu) and zinc (Zn) in acid mine drainage (AMD) after limestone treatment are worth studying to better understand the impacts of limestone AMD treatment. In this study, the wastewater from a copper mine ditch was sampled. Two different doses of limestone (S: 5.33 g L-1 and SS: 8.00 g L-1) were applied to adjust the pH range of the sampled AMD. The concentrations of Fe, Cu and Zn in the supernatant and the levels of iron (oxyhydr)oxides and heavy metals in AMD sediments were dynamically monitored for 300 days to analyze the transformation of the secondary iron mineral phase and the role iron (oxyhydr)oxides play in the removal and stabilization of Cu and Zn. The results showed that the pH rose rapidly to 6.82, decreased to 5.82 on the 150th day, and finally decreased to approximately 4.63 by the 300th day, when the dosage of limestone (S) was 5.33 g L-1. Goethite was the main form of iron oxides in the sediments. As the incubation time increased, so did the content of crystalline Fe (oxyhydr)oxides. In addition, the Cu and Zn content in the fraction of crystalline Fe (oxyhydr)oxides increased as the corresponding iron (oxyhydr)oxide increased. When the high dosage of limestone (8.00 g L-1 or SS) was applied, the pH remained at approximately at 7.46 during the whole period and goethite and lepidocrocite were present in the sediment. Amorphous/ poorly crystalline Fe-oxyhydroxide was the main product after SS limestone dosage, indicating that the risk of Cu and Zn reactivation in the sediment was higher with a higher limestone treatment dosage.

Carbon dots as light-responsive oxidase-like nanozyme for colorimetric detection of total antioxidant capacity in fruits.

Evaluation of total antioxidant capacity (TAC) in fruits is essential for dietary guidance and health monitoring. Here, we have exploited light-response carbon dots (CDs) as oxidase-like nanozyme to determine the TAC of fruits. The CDs possess excellent oxidase-like activity with light stimulation due to the accelerated intramolecular charge transfer caused by abundant electron donating/drawing groups in precursors. The scavenger experiment reveals that the catalytic intermediate could be hydroxyl radical, which can oxidize the colorimetric substrate. With the introduction of antioxidants, the oxidization of colorimetric substrate will be alleviated due to the scavenging of this intermediate by antioxidants. Based on this, we have successfully detected three antioxidants and obtained TAC of fruits with desirable results. This work affords a rapid, cost-effective and convenient analysis tool for TAC, as well as building a strong bridge between CDs and the development of photo-responsive oxidase-like nanozymes.

Research on the Corrosion Behavior of Q235 Pipeline Steel in an Atmospheric Environment through Experiment.

Low-carbon steel pipelines are frequently used as transport pipelines for various media. As the pipeline transport industry continues to develop in extreme directions, such as high efficiency, long life, and large pipe diameters, the issue of pipeline reliability is becoming increasingly prominent. This study selected Q235 steel, a typical material for low-carbon steel pipelines, as the research object. In accordance with the pipeline service environment and the accelerated corrosion environment test spectrum, cyclic salt spray accelerated corrosion tests that simulated the effects of the marine atmosphere were designed and implemented. Corrosion properties, such as corrosion weight loss, morphology, and product composition of samples with different cycles, were characterized through appearance inspection, scanning electron microscopy analysis, and energy spectrum analysis. The corrosion behavior and mechanism of Q235 low-carbon steel in the enhanced corrosion environment were studied, and the corrosion weight loss kinetics of Q235 steel was verified to conform to the power function law. During the corrosion process, the passivation film on the surface of the low-carbon steel and the dense and stable α-FeOOH layer formed after the passivation film was peeled off played a role in corrosion resistance. The passivation effect, service life, and service limit of Q235 steel were studied and determined, and an evaluation model for quick evaluation of the corrosion life of Q235 low-carbon steel was established. This work provides technical support to improve the life and reliability of low-carbon steel pipelines. It also offers a theoretical basis for further research on the similitude and relevance of cyclic salt spray accelerated corrosion testing.

Quantitative parameters obtained from gadobenate dimeglumine-enhanced MRI at the hepatobiliary phase can predict post-hepatectomy liver failure and overall survival in patients with hepatocellular carcinoma.

PURPOSE: To determine the value of the quantitative parameters obtained from gadobenate dimeglumine-enhanced magnetic resonance imaging (MRI) at the hepatobiliary phase for predicting post-hepatectomy liver failure and overall survival in patients with hepatocellular carcinoma. METHOD: This multicenter retrospective study included 307 patients who underwent gadobenate dimeglumine-enhanced MRI. The quantitative liver-to-portal vein contrast ratio (LPC) and liver-spleen contrast ratio (LSC) at the hepatobiliary phase were measured. Logistic regression analyses were used to evaluate risk factors for post-hepatectomy liver failure. The capacity of the LPC and LSC to predict post-hepatectomy liver failure was evaluated via receiver operating characteristic (ROC) curve. The Cox proportional hazards regression was used to identify prognostic factors for overall survival (OS). RESULTS: Post-hepatectomy liver failure was observed in 69 patients (22.5%). The LPC and LSC were independent risk factors for the development of post-hepatectomy liver failure, and the areas under the ROC curves of LPC and LSC were 0.882 and 0.782, respectively. The predictive performance of LPC for post-hepatectomy liver failure was superior to LSC. The LPC and LSC were also significant prognostic factors for OS. The cut-off values for the LPC and LSC were 1.07 and 0.89, respectively. The 5-year OS rate was higher in patients with LPC > 1.07 or LSC > 0.89 than in patients with LPC ≤ 1.07 or LSC ≤ 0.89. CONCLUSIONS: The quantitative parameters obtained from gadobenate dimeglumine-enhanced MRI at the hepatobiliary phase were effective imaging biomarkers for predicting both post-hepatectomy liver failure and overall survival in patients with hepatocellular carcinoma.

Response boosting-based approach for absolute quantification of gelatin peptides using LC-MS/MS.

Response-boosting of MS signal was observed in gelatin samples due to abundant Glycine residues produced by collagen enzymolysis. In this work, a new strategy utilizing response-boosting to enhance detection sensitivity was developed for absolute quantification of Asini Corii Colla, a kind of gelatin commonly used as food therapy products in Asia, by high performance liquid chromatography coupled to tandem mass spectrometry. Peptidomics analysis was used to evaluate the similarity between eight different protein matrices, and deer-hide gelatin was selected as the appropriate simulated matrix. Isotope-labelled internal standard was used to compensate the matrix effect and construct matrix-matched calibration curves. The established method showed reliability in absolute quantification of three species-specific gelatin peptides with good linearity (r2 > 0.997), precision (RSD < 8.5%), repeatability (RSD < 8.9%), accuracy (recovery 89.4%∼106.5%) and sensitivity (LOD 0.02 âˆ¼ 0.98 ng/mL). Thus, the present response-boosting based protocol provides a promising application in quality control of food rich in gelatins.

Discovery of peptide biomarkers by label-free peptidomics for discrimination of horn gelatin and hide gelatin from Cervus nippon Temminck.

Gelatin and gelatin-based derivatives have been attracting worldwide attention as health-food ingredients. Deer horn gelatin (DCG), a well-known and expensive gelatin food in Asia, has suffered adulterants by adding deer-hide gelatin (DHG) in it. However, robust and effective methods which could differentiate DCG from DHG are still unavailable. This study is committed to discover peptide biomarkers to distinguish DCG from DHG using label-free peptidomics by nanoLC-MS/MS. Multivariate statistical analysis combined with glycosylation sites analysis of peptides was applied to visualize the difference between DCG and DHG. As a result, four peptide biomarkers for distinguishing DCG and DHG were confirmed and validated by UPLC-MS/MS and MRM mode, which was also used to calculate adulteration percentage in commercial samples. The presented strategy may be also particularly helpful in the in-depth authentication of food gelatins from different tissues of the same species.

Reduced kinase D­interacting substrate of 220 kDa (Kidins220) in pancreatic cancer promotes EGFR/ERK signalling and disease progression.

Kidins220 is a transmembrane scaffold protein involved in several types of cancer. The aim of the present study was to examine the role of Kidins220 in tumorigenesis and disease progression of pancreatic cancer. The relevant signalling pathways including EGFR, EMT, and MMP were also investigated. The expression of Kidins220 was examined at the transcript and protein level. The Kidins220 knockdown cell model was established and its influence on cellular functions was determined. Involvement of Kidins220 in tumorigenesis and metastasis was examined in CD1 mice, respectively. The results showed that, reduced Kidin220 expression was associated with tumorigenesis, metastasis, and overall survival of pancreatic cancer. Knockdown of Kidins220 promoted proliferation, colony formation and tumorigenic capacity of pancreatic cancer cells in vitro and in vivo, respectively. Kidins220 regulated pancreatic cancer cell migration through the EGFR/AKT/ERK signalling pathway. Furthermore, enhanced EMT was observed in the pancreatic cancer cell lines with the knockdown of Kidins220, underlying EGFR regulation. Kidins220 also affected cell invasion via MMP1. A reduced expression of Kidins220 was observed in pancreatic cancer, which is associated with disease progression, distant metastasis and poor prognosis. The loss of Kidins220 in pancreatic cancer may contribute to disease progression through the upregulation of EGFR and downstream signalling.

Noggin is associated with a poor prognosis of gastric cancer by promoting the proliferation of gastric cancer cells via the upregulation of EGFR.

Noggin is an antagonist of bone morphogenetic proteins (BMP), being indispensable for certain developmental events. The present study aimed to examine the role of Noggin in the development and prognosis of gastric cancer (GC) and to elucidate the underlying mechanisms. The expression of Noggin in GC was evaluated by RT­qPCR, immunohistochemistry and by the analyses of publicly available databases. The effects of Noggin on proliferation, cell cycle, adhesion, invasion, colony formation and tumour spheroid were examined following both the knockdown and overexpression of Noggin in GC cell lines. The involvement of epidermal growth factor receptor (EGFR) signalling was examined by western blot analysis and by using small molecule inhibitors. As a result, a higher expression of Noggin in GC was found to be associated with a poorer overall survival. Noggin overexpression promoted the proliferation and colony formation of GC cells by promoting cell cycle progression. The knockdown of Noggin in HGC27 cells exerted an opposite effect on proliferation, colony formation and cell cycle progression. Noggin expression positively correlated with EGFR expression in both GC cell line models and The Cancer Genome Atlas human GC cohort. Targeting EGFR and its downstream pathways diminished cell proliferation which was promoted by Noggin. Furthermore, Noggin overexpression resulted in an enhanced nuclear translocation of ß­catenin, leading to an upregulation of EGFR. Thus, the findings of the present study demonstrate that Noggin promotes the proliferation of GC cells by upregulating EGFR and enhancing a vicious circle formed by ß­catenin, EGFR, ERK and Akt.

Bone morphogenetic proteins mediate crosstalk between cancer cells and the tumour microenvironment at primary tumours and metastases (Review).

Bone morphogenetic proteins (BMP) are pluripotent molecules, co­ordinating cellular functions from early embryonic and postnatal development to tissue repair, regeneration and homeostasis. They are also involved in tumourigenesis, disease progression and the metastasis of various solid tumours. Emerging evidence has indicated that BMPs are able to promote disease progression and metastasis by orchestrating communication between cancer cells and the surrounding microenvironment. The interactions occur between BMPs and epidermal growth factor receptor, hepatocyte growth factor, fibroblast growth factor, vascular endothelial growth factor and extracellular matrix components. Overall, these interactions co­ordinate the cellular functions of tumour cells and other types of cell in the tumour to promote the growth of the primary tumour, local invasion, angiogenesis and metastasis, and the establishment and survival of cancer cells in the metastatic niche. Therefore, the present study aimed to provide an informative summary of the involvement of BMPs in the tumour microenvironment.

Increased Expression of Gremlin1 Promotes Proliferation and Epithelial Mesenchymal Transition in Gastric Cancer Cells and Correlates With Poor Prognosis of Patients With Gastric Cancer.

BACKGROUND/AIM: Gremlin1 (GREM1) plays an important role in certain malignancies by antagonising bone morphogenetic proteins and regulating angiogenesis directly/indirectly. The present study aimed to investigate the role of Gremlin1 in the development and progression of gastric cancer (GC). MATERIALS AND METHODS: Expression of GREM1 in GCs was examined using quantitative real time PCR and The Cancer Genomic Atlas (TCGA) data. Influence on cellular functions was determined in both Gremlin1 knockdown and overexpression cell line models. RESULTS: GREM1 expression was up-regulated in GCs, which was correlated with poorer survival. Increased GREM1 expression was significantly correlated with tumour growth/invasion and lymphatic metastasis. Gremlin1 promoted proliferation and tumourigenic capacity of GC cells in vitro. GREM1 expression was associated with epithelial mesenchymal transition (EMT), angiogenesis and lymphangiogenesis in GC. CONCLUSION: Increased GREM1 expression in GCs is associated with disease progression and poor prognosis in which EMT, angiogenesis and lymphangiogenesis are likely involved.

Acupuncture as a multifunctional neuroprotective therapy ameliorates cognitive impairment in a rat model of vascular dementia: A quantitative iTRAQ proteomics study.

AIMS: Acupuncture has been reported to affect vascular dementia through a variety of molecular mechanisms. An isobaric tag for relative and absolute quantification (iTRAQ) with high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses makes it possible to attain a global profile of proteins. Hence, we used an iTRAQ-LC-MS/MS strategy to unravel the underlying mechanism of acupuncture. METHODS: Wistar rats were subjected to vascular dementia with bilateral common carotid occlusion. Acupuncture was intervened for 2 weeks at 3 days after surgery. The Morris water maze was used to assess the cognitive function. Proteins were screened by quantitative proteomics and analyzed by bioinformatic analysis. Four differentially expressed proteins (DEPs) were validated by western blot. The reactive oxygen species (ROS) production, neuron cell loss, and long-term potentiation (LTP) were determined after western blot. RESULTS: Acupuncture at proper acupoints significantly improved cognitive function. A total of 31 proteins were considered DEPs. Gene ontology (GO) analysis showed that most of the DEPs were related to oxidative stress, apoptosis, and synaptic function, which were regarded as the major cellular processes related to acupuncture effect. Western blot results confirm the credibility of iTRAQ results. Acupuncture could decrease ROS production, increase neural cell survival, and improve LTP, which verified the three major cellular processes. CONCLUSION: Acupuncture may serve as a promising clinical candidate for the treatment of vascular dementia via regulating oxidative stress, apoptosis, or synaptic functions.

Kidins220 and tumour development: Insights into a complexity of cross-talk among signalling pathways (Review).

The mechanistic complexes of kinase D-interacting substrate of 220 kDa/ankyrin repeat-rich membrane spanning (Kidins220/ARMS) bind and integrate a variety of cellular cues to mediate neuronal activities such as neuronal differentiation, survival, and cytoskeleton remodelling by interacting with a variety of binding partners. Accumulated evidence has also indicated its role in the regulation of vascular development. Mice with Kidins220 knockdown phenotypically present with cardiovascular abnormalities. Kidins220 also contributes to immunomodulation in combination with B cells and T cells. Moreover, emerging evidence has revealed that this protein regulates many crucial cellular processes and thus has been implicated in an increasing number of malignancies. Here, we review recent advances in our understanding of Kidins220 and its role in cancer development. Further investigation is warranted to shed light on the role played by Kidins220 in the dynamic arrangement of the cytoskeleton and epithelial-mesenchymal transition, and its implication in tumourigenesis and cancer progression.

Psoriasin promotes invasion, aggregation and survival of pancreatic cancer cells; association with disease progression.

Psoriasin (S100A7) is an 11-kDa small calcium binding protein initially isolated from psoriatic skin lesions. It belongs to the S100 family of proteins which play an important role in a range of cell functions including proliferation, differentiation, migration and apoptosis. Aberrant Psoriasin expression has been implicated in a range of cancers and is often associated with poor prognosis. This study examined the role of Psoriasin on pancreatic cancer cell functions and the implication in progression of the disease. Expression of Psoriasin was determined in a cohort of pancreatic tissues comprised of 126 pancreatic tumours and 114 adjacent non-tumour pancreatic tissues. Knockdown and overexpression of Psoriasin in pancreatic cancer cells was performed using specifically constructed plasmids, which either had anti-Psoriasin ribozyme transgene or the full length human Psoriasin coding sequence. Psoriasin knockdown and overexpression was verified using conventional RT-PCR and qPCR. The effect of manipulating Psoriasin expression on pancreatic cancer cell functions was assessed using several in vitro cell function assays. Local invasive pancreatic cancers extended beyond the pancreas expressed higher levels of Psoriasin transcripts compared with the cancers confined to the pancreas. Primary tumours with distant metastases exhibited a reduced expression of Psoriasin. Psoriasin overexpression cell lines exhibited significantly increased growth and migration compared to control cells. In addition, Psoriasin overexpression resulted in increased pancreatic cancer cell invasion which was associated with upregulation of matrix metalloproteinase-2 (MMP-2) and MMP-9. Overexpression of Psoriasin also promoted aggregation and survival of pancreatic cancer cells when they lost anchorage. Taken together, higher expression of Psoriasin was associated with local invasion in pancreatic cancers. Psoriasin expression is associated with pancreatic cancer cell growth, migration, cell-matrix adhesion, and invasion via regulation of MMPs. As such, the proposed implications of Psoriasin in invasion, disease progression and as a potential therapeutic target warrant further investigation.

Expression of phospholipase C isozymes in human breast cancer and their clinical significance.

Phospholipase C (PLC) regulates a number of cellular behaviours including cell motility, cell transformation, differentiation and cell growth. PLC plays a regulatory role in cancer cells partly by acting as signalling intermediates for cytokines such as EGF and interleukins. The current study examined the expression of the PLC isozymes in human breast cancer and corresponding clinical relevance. Transcript levels of human PLC-α, -ß1, -δ, -ε, and -γ1 in human breast cancer tissues were quantitatively determined by real-time PCR. Immunochemical staining was performed for PLC-δ. The clinical relevance was analysed with clinic pathological information. Mammary tissues widely expressed PLC-α, -ß1, -δ, -ε, and -γ1. Significantly high levels of PLC -ß1 and -ε were seen in breast cancer tissues in comparison with normal mammary gland tissues. PLC-γ1 however, showed marginally low levels in tumour tissues. No significant difference was seen in the expression of the PLC isozymes in tumours with lymph node metastases. Moderately and poorly differentiated breast tumours (grade 2 and grade 3) had significantly higher levels of PLC-γ1, compared with well differentiated tumours. High levels of PLC-δ were significantly correlated with a shorter disease-free survival. The altered expression of other isozymes had no correlation with the survival. It is concluded that mammary tissues differentially expressed PLC isozymes. These isozymes have certain implications in the disease development and progression, with PLC-δ showing a significant correlation with shorter disease-free survival.