Preference and regulation mechanism mediated via mobile genetic elements for antibiotic and metal resistomes during composting amended with nano ZVI loaded on biochar.

This study assessed the effectiveness of nano zero-valent iron loaded on biochar (BC-nZVI) during swine manure composting. BC-nZVI significantly reduced the abundance of antibiotic resistance genes (ARGs), metal resistance genes (MRGs), and mobile genetic elements (MGEs). BC-nZVI modified the preference of MGEs to carry ARGs and MRGs, and the corrosion products of BC-nZVI could destroy cell structure, hinder electron transfer between cells, and weaken the association between ARGs, MRGs, and host bacteria. Functional genes analysis revealed that BC-nZVI down-regulated the abundance of genes affecting the transmission and metabolism of ARGs and MRGs, including type IV secretion systems, transporter systems, two-component systems, and multidrug efflux pumps. Furthermore, the BC-nZVI decreased genes related to flagella and pili production and cell membrane permeability, thereby hindering the transfer of ARGs, MRGs, and MGEs in the environment. Redundancy analysis demonstrated that changes in the microbial community induced by BC-nZVI were pivotal factors impacting the abundance of ARGs, MRGs, and MGEs. Overall, this study confirmed the efficacy of BC-nZVI in reducing resistance genes during swine manure composting, offering a promising environmental strategy to mitigate the dissemination of these contaminants.

Structural biology of terpene synthases.

Structural biology research of terpene synthases (TSs) has provided a useful basis to understand their catalytic mechanisms in producing diverse terpene products with polycyclic ring systems and multiple chiral centers. However, compared to the large numbers of>95,000 terpenoids discovered to date, few structures of TSs have been solved and the understanding of their catalytic mechanisms is lagging. We here (i) introduce the basic catalytic logic, the structural architectures, and the metal-binding conserved motifs of TSs; (ii) provide detailed experimental procedures, in gene cloning and plasmid construction, protein purification, crystallization, X-ray diffraction data collection and structural elucidation, for structural biology research of TSs; and (iii) discuss the prospects of structure-based engineering and de novo design of TSs in generating valuable terpene molecules, which cannot be easily achieved by chemical synthesis.

Measuring Heat Dissipation and Entropic Potential in Battery Cathodes Made with Conjugated and Conventional Polymer Binders Using Operando Calorimetry.

This study explores the influence of electronic and ionic conductivities on the behavior of conjugated polymer binders through the measurement of entropic potential and heat generation in an operating lithium-ion battery. Specifically, the traditional poly(vinylidene fluoride) (PVDF) binder in LiNi0.8Co0.15Al0.05O2 (NCA) cathode electrodes was replaced with semiconducting polymer binders based on poly(3,4-propylenedioxythiophene). Two conjugated polymers were explored: one is a homopolymer with all aliphatic side chains, and the other is a copolymer with both aliphatic and ethylene oxide side chains. We have shown previously that both polymers have high electronic conductivity in the potential range of NCA redox, but the copolymer has a higher ionic conductivity and a slightly lower electronic conductivity. Entropic potential measurements during battery cycling revealed consistent trends during delithiation for all of the binders, indicating that the binders did not modify the expected NCA solid solution deintercalation process. The entropic signature of polymer doping to form the conductive state could be clearly observed at potentials below NCA oxidation, however. Operando isothermal calorimetric measurements showed that the conductive binders resulted in less Joule heating compared to PVDF and that the net electrical energy was entirely dissipated as heat. In a comparison of the two conjugated polymer binders, the heat dissipation was lower for the homopolymer binder at lower C-rates, suggesting that electronic conductivity rather than ionic conductivity was the most important for reducing Joule heating at lower rates, but that ionic conductivity became more important at higher rates.

Multi-level, forming and filament free, bulk switching trilayer RRAM for neuromorphic computing at the edge.

CMOS-RRAM integration holds great promise for low energy and high throughput neuromorphic computing. However, most RRAM technologies relying on filamentary switching suffer from variations and noise, leading to computational accuracy loss, increased energy consumption, and overhead by expensive program and verify schemes. We developed a filament-free, bulk switching RRAM technology to address these challenges. We systematically engineered a trilayer metal-oxide stack and investigated the switching characteristics of RRAM with varying thicknesses and oxygen vacancy distributions to achieve reliable bulk switching without any filament formation. We demonstrated bulk switching at megaohm regime with high current nonlinearity, up to 100 levels without compliance current. We developed a neuromorphic compute-in-memory platform and showcased edge computing by implementing a spiking neural network for an autonomous navigation/racing task. Our work addresses challenges posed by existing RRAM technologies and paves the way for neuromorphic computing at the edge under strict size, weight, and power constraints.

Research on the decomposition mechanisms of lithium silicate ores with different crystal structures by autotrophic and heterotrophic bacteria.

Ores serve as energy and nutrient sources for microorganisms. Through complex biochemical processes, microorganisms disrupt the surface structure of ores and release metal elements. However, there is limited research on the mechanisms by which bacteria with different nutritional modes act during the leaching process of different crystal structure ores. This study evaluated the leaching efficiency of two types of bacteria with different nutritional modes, heterotrophic bacterium Bacillus mucilaginosus (BM) and autotrophic bacterium Acidithiobacillus ferrooxidans (AF), on different crystal structure lithium silicate ores (chain spodumene, layered lepidolite and ring elbaite). The aim was to understand the behavioral differences and decomposition mechanisms of bacteria with different nutritional modes in the process of breaking down distorted crystal lattices of ores. The results revealed that heterotrophic bacterium BM primarily relied on passive processes such as bacterial adsorption, organic acid corrosion, and the complexation of small organic acids and large molecular polymers with metal ions. Autotrophic bacterium AF, in addition to exhibiting stronger passive processes such as organic acid corrosion and complexation, also utilized an active transfer process on the cell surface to oxidize Fe2+ in the ores for energy maintenance and intensified the destruction of ore lattices. As a result, strain AF exhibited a greater leaching effect on the ores compared to strain BM. Regarding the three crystal structure ores, their different stacking modes and proportions of elements led to significant differences in structural stability, with the leaching effect being highest for layered structure, followed by chain structure, and then ring structure. These findings indicate that bacteria with different nutritional modes exhibit distinct physiological behaviors related to their nutritional and energy requirements, ultimately resulting in different sequences and mechanisms of metal ion release from ores after lattice damage.

Sensitive Electrochemical Detection of Carcinoembryonic Antigen Based on Biofunctionalized Nanochannel Modified Carbonaceous Electrode.

The convenient construction of carbon-based electrochemical immunosensors with high performance is highly desirable for the efficient detection of tumor biomarkers. In this work, an electrochemical immunosensor was fabricated by integrating a biofunctionalized mesoporous silica nanochannel film with a carbon-based electrode, which can enable the sensitive determination of carcinoembryonic antigen (CEA) in serum. The commonly used carbonaceous electrode, glassy carbon electrode (GCE), was employed as the supporting electrode and was pre-treated through electrochemical polarization to achieve the stable binding of a vertically ordered mesoporous silica film with amino groups (NH2-VMSF) without the use of any adhesive layer. To fabricate the immunorecognition interface, antibodies were covalently immobilized after the amino groups on the outer surface of NH2-VMSF was derivatized to aldehyde groups. The presence of amino sites within the high-density nanochannels of NH2-VMSF can facilitate the migration of negatively charged redox probes (Fe(CN)63-/4-) to the supporting electrode through electrostatic adsorption, leading to the generation of electrochemical signals. In the presence of CEA, the formation of immunocomplexes on the recognitive interface can reduce the electrochemical signal of Fe(CN)63-/4- on the supporting electrode. Based on this principle, the sensitive electrochemical detection of CEA was achieved. CEA can be determined to range from 0.01 ng mL-1 to 100 ng mL-1 with a limit of detection of 6.3 pg mL-1. The fabricated immunosensor exhibited high selectivity, and the detection of CEA in fetal bovine serum was achieved.

Comparison of manual sutures and laparoscopic stapler for pancreatic stump closure techniques in robotic distal pancreatectomy: a single-center experience.

BACKGROUND: Postoperative pancreatic fistulas (POPFs) are prevalent and major postoperative complications of distal pancreatectomy (DP). There are numerous ways to manage the pancreatic stump. However, no single approach has been shown to be consistently superior. Moreover, the potential role of robotic systems in reducing POPFs has received little attention. METHODS: The clinical data of 119 patients who had consecutively received robotic distal pancreatectomy between January 2019 and December 2022 were retrospectively analyzed. Patients were divided into two groups according to the method of handling the pancreatic stump. The attributes of the patients and the variables during the perioperative period were compared. RESULTS: The analysis included 72 manual sutures and 47 stapler procedures. The manual suture group had a shorter operative time (removing installation time) than the stapler group (125.25 ± 63.04 min vs 153.30 ± 62.03 min, p = 0.019). Additionally, the manual suture group had lower estimated blood loss (50 mL vs 100 mL, p = 0.009) and a shorter postoperative hospital stay. There were no significant differences in the incidence of clinically relevant POPFs between the two groups (18.1% vs 23.4%, P > 0.05). No perioperative death occurred in either group. CONCLUSION: The manual suturing technique was shown to have an incidence of POPFs similar to the stapler technique in robotic distal pancreatectomy and to be safe and feasible.

Fe3 C/nanocarbon-Enabled Lithium Dendrite Mitigation in Lithium-Sulfur batteries.

Lithium dendrite-induced short circuits and material loss are two major obstacles to the commercialization of lithium-sulfur (Li-S) batteries. Here, a nanocarbon composite consisting of cotton-derived Fe3 C-encapsulated multiwalled carbon nanotubes (Fe3 C-MWCNTs) and graphene effectively traps polysulfides to suppress lithium dendrite growth is reported. Machine learning combined with molecular dynamics (MD) simulations unveils a new polysulfide-induced lithium dendrite formation mechanism: the migration of polysulfides away from the anode drags out lithium protrusions through localized lattice distortion of the lithium anode and traps lithium ions in the surrounding electrolyte, leading to lithium dendrite formation. The Li-S battery, constructed using the composite of cotton-derived Fe3 C-MWCNTs and graphene that serves as both the sulfur host and the anode interlayer, exhibits exceptional cycling stability, impressive capacity retention, and effective mitigation of lithium dendrite formation. The findings offer valuable strategies to prevent lithium dendrite formation and enhance understanding of lithium dendrite growth in Li-S batteries.

Electrostatic Nanocage-Confined Probe for Electrochemical Detection of CA19-9 in Human Serum.

Prompt and accurate detection of CA19-9 in human serum has great clinical significance for the early diagnosis and disease monitoring of cancer. Herein, we develop a convenient and antifouling electrochemical sensor for CA19-9 determination by immobilization of both an electrochemical redox probe [methylene blue (MB)] and immunorecognition element (CA19-9 antibody) on an electrostatic nanocage consisting of bipolar silica nanochannel array (bp-SNA). bp-SNA is composed of a negatively charged inner layer (n-SNA) and positively charged outer layer (p-SNA), which could be stably prepared on indium tin oxide (ITO) in several seconds using a two-step electrochemically assisted self-assembly approach and display asymmetric surface charges for confinement and enrichment of cationic MB into the inner n-SNA layer through electrostatic interaction. Modification of the CA19-9 antibody on the top surface of bp-SNA confers the sensing interface with specific recognition capacity. An antibody-antigen complex formed at the as-prepared immunosensor causes the decreased electrochemical signals of MB, achieving sensitive determination of CA19-9 with a wider linear dynamic range from 10 µU/mL to 50 U/mL and a low detection limit (3 µU/mL). Furthermore, accurate and feasible analysis of the CA19-9 amount in human serum samples by our proposed probe-integrated electrochemical immunosensor is realized.

Lithium extraction from typical lithium silicate ores by two bacteria with different metabolic characteristics: Experiments, mechanism and significance.

Microorganisms obtain inorganic nutrients or energy from specific minerals to selectively weather minerals, but few studies on the differences in metabolic components of different functional bacteria lead to different weathering effects. This study evaluated the leaching effects of two bacteria with distinct metabolic characteristics on lithium silicate minerals with different structures. We aimed to understand the microscopic mechanism of crystal destruction of lithium silicate minerals with different structures under the action of microorganisms. The results showed that the metabolites produced by an acid producing silicate strain Raoultella sp. Z107 (strain Z107) had a high content of organic acids, among which lactic acid was up to about 11 g/L. Bacillus mucilaginosus 21,699 (strain BM) secreted capsular polysaccharide with a high content of 14.84 mg/L. The metabolic activities of the two strains were significantly different. Through the analysis of the leaching residue, it was found that the lithium silicate minerals were acid etched, interlayer domains expanded, crystallinity decreased, and metal bonds were broken under the action of bacteria. The dissolution of lithium silicate minerals by bacteria is a combination of bacterial adsorption, organic acid corrosion, and complexation of small molecular organic acids and macromolecular polymers with metal ions. The acid erosion and complexation effects of organic acids are greater than the single complexation of capsular polysaccharides, and the layered lepidolite is more likely to be decomposed by the weathering of bacterial metabolites than the chain structure spodumene. These results indicate that the diversity of metabolic activity of bacteria from different sources and the sequence and decomposition mechanism of metal ions released from minerals after lattice destruction are also different. Microorganisms decompose minerals for energy and nutrients, and eventually become the main players in the transformation of elements in biogeology.

[Microbial Community Composition and Diversity in Metal Sulfide Mine Water Systems].

To understand the characteristics of heavy metal pollution and physicochemical properties caused by mining activities in mine water systems and the response of the microbial community to habitats with different contamination levels, this study selected different types of water (mining area wastewater, spoil heap area wastewater, dressing area wastewater, mine seepage water, and pond water) as the variables related to the mining activities in the water system of the Shizishan mining area in Tongling, Anhui Province. The pollution characteristics and physicochemical properties were compared, and the relationship between environmental factors and the microbial communities were analyzed. The results showed that the content of heavy metals, the physicochemical properties, and the structure and diversity of the microbial community of different types of water were significantly different in different mine areas, among which the most seriously polluted areas were the mining area, the spoil heap area, and the dressing area. There were significant differences in microbial community structure among different functional types of wastewaters, and the diversity and abundance of the microbial community in DW with the heaviest heavy metal pollution were weaker than those in the other four regions. PcoA analysis showed that samples of similar water types had similar clustering. Spearman correlation heat map analysis and canonical correlation analysis (CCA) indicated that heavy metal pollution, pH, electrical conductivity (EC), SO2-4, and chemical oxygen demand (COD) had the greatest effect on the microbial communities in the mine water systems. Moreover, this study found that Proteobacteria, Euryarchaeota, and Bacteroidetes dominated in mine water systems, and their potential use could be explored in the future. Our results provide a better understanding of the different types of water pollution characteristics in mine water systems and the key factors that determine the microbial community structure.

Antibiotic resistance gene profiles and evolutions in composting regulated by reactive oxygen species generated via nano ZVI loaded on biochar.

In this study, nano zero-valent iron loaded on biochar (BC-nZVI) was analyzed for its effects on antibiotic resistance genes (ARGs) in composting. The results showed that BC-nZVI increased reactive oxygen species (ROS) production, and the peak values of H2O2 and OH were 22.95 % and 55.30 % higher than those of the control group, respectively. After 65 days, the relative abundances of representative ARGs decreased by 56.12 % in the nZVI group (with BC-nZVI added). An analysis of bacterial communities and networks revealed that Actinobacteria, Proteobacteria, and Firmicutes were the main hosts for ARGs, and BC-nZVI weakened the link between ARGs and host bacteria. Distance-based redundancy analysis showed that BC-nZVI altered the microbial community structure through environmental factors and that most ARGs were negatively correlated with ROS, suggesting that ROS significantly affected the relative abundance of ARGs. According to these results, BC-nZVI showed potential for decreasing the relative abundance of ARGs in composting.

Reoperation for heterochronic intraductal papillary mucinous neoplasm of the pancreas after bile duct neoplasm resection: A case report.

BACKGROUND: Intraductal papillary neoplasm of the bile duct (IPNB) and intraductal papillary mucinous neoplasm (IPMN) of the pancreas have similar pathological manifestations. However, they often develop separately and it is rare for both to occur together. Patients presenting with heterochronic IPMN after IPNB are prone to be misdiagnosed with tumor recurrence. CASE SUMMARY: A 67-year-old male patient was admitted 8.5 years after IPNB carcinoma and 4 years after the discovery of a pancreatic tumor. A left hepatic bile duct tumor with distal bile duct dilatation was found 8.5 years ago by the computed tomography; therefore, a left hepatectomy was performed. The postoperative pathological diagnosis was malignant IPNB with negative cutting edge and pathological stage T1N0M0. Magnetic resonance imaging 4 years ago showed cystic lesions in the pancreatic head with pancreatic duct dilatation, and carcinoembryonic antigen continued to increase. Positron emission tomography showed a maximum standard uptake value of 11.8 in the soft tissue mass in the pancreatic head, and a malignant tumor was considered. Radical pancreatoduodenectomy was performed. Postoperative pathological diagnosis was pancreatic head IPMN with negative cutting edge, pancreaticobiliary type, stage T3N0M0. He was discharged 15 d after the operation. Follow-up for 6 mo showed no tumor recurrence, and quality of life was good. CONCLUSION: IPNB and IPMN are precancerous lesions with similar pathological characteristics and require active surgery and long-term follow-up.

A potential heavy metals detoxification system in composting: Biotic and abiotic synergy mediated by shell powder.

Regulating heavy metal resistance genes (HMRGs) was an effective method for heavy metal resistant bacteria (HMRB) to cope with heavy metal stress during dairy manure composting. This research aimed to investigate heavy metal detoxification mediated by shell powder (SP) in composting and the response of HMRB and HMRGs to changes in heavy metal bioavailability during composting. Research showed that SP additive reduced the bioavailability of Zu, Cu, and Mn by 10.64%, 13.90% and 14.14%, respectively. SP increased the composition percentage of humic acid (HA) in humus (HS) by 8%. SP enhanced the resistance of Actinobacteria to heavy metals and improved the regulation of HMRGs. Correlation analysis demonstrated that the bioavailability of heavy metals was positively correlated with most HMRGs. HA was significantly negatively correlated with the bioavailability of Zn, Cu and Mn. Therefore, SP additive could be a novel strategy for heavy metals detoxification during composting.

The removal performances and evaluation of heavy metals, antibiotics, and resistomes driven by peroxydisulfate amendment during composting.

This study aimed to explore the effect of peroxydisulfate on the removal of heavy metals, antibiotics, heavy metal resistance genes (HMRGs), and antibiotic resistance genes (ARGs) during composting. The results showed that peroxydisulfate achieved the passivation of Fe, Mn, Zn, and Cu by promoting their speciation variations, thus reducing their bioavailability. And the residual antibiotics were better degraded by peroxydisulfate. In addition, metagenomics analysis indicated that the relative abundance of most HMRGs, ARGs, and MGEs was more effectively down-regulated by peroxydisulfate. Network analysis confirmed Thermobifida and Streptomyces were dominant potential host bacteria of HMRGs and ARGs, whose relative abundance was also effectively down-regulated by peroxydisulfate. Finally, mantel test showed the significant effect of the evolution of microbial communities and strong oxidation of peroxydisulfate on the removal of pollutants. These results suggested that heavy metals, antibiotics, HMRGs, and ARGs shared a joint fate of being removed driven by peroxydisulfate during composting.

Iron reduction in composting environment synergized with quinone redox cycling drives humification and free radical production from humic substances.

The aim of this paper was to investigate the influence of Fe (III) on humification and free radicals evolution. The experimental data showed that the experimental group (CT) with Fe2(SO4)3 had a better degree of humification than the control group (CK). The humic substances (HS) content was 10% higher in CT (23.94 mg·g-1) than in CK (21.54 mg·g-1) in the final. Fe (III) contributed significantly to the formation of free radicals in HS. The amount of H2O2 in CT increased to 74.8 mmol·kg-1, while CK was only 46.5 mmol·kg-1. The content of semiquinone free radical was 10.32 × 1011 spins/mm3 in CT, 5.11 × 1011 spins/mm3 in CK in the end. Several iron-reducing bacteria were detected in composting, among which Paenibacillus was dominant. The above findings suggested that the application of Fe2(SO4)3 enhanced the iron reduction synergistic quinone redox cycling and promoted the generation of free radicals during the humification of composting.

Ear reconstruction stage I: Minor modifications in sculpting the auricle support using the 7th and 8th costal cartilages.

BACKGROUND: The verisimilitude of the reconstructed auricle and its long-term stability largely depends on the framework sculpting. This study described three kinds of minor modifications based on Firmin's way of sculpting the auricle framework and reported the clinical outcomes achieved with them. METHODS: We conducted a retrospective study of congenital microtia patients undergoing detail-improved auricular reconstruction from June 2016 to June 2020. The three kinds of minor modifications included: (1) fabricating the base frame using the 7th costal cartilage, (2) fabricating the helix and the antihelix complex using the 8th costal cartilage, and (3) fabricating the helix using the combination of the 8th and 9th costal cartilage. RESULTS: Ninety-eight patients (aged 9-27 years, 62.2% male) were included. Ninety-five patients (97.0%) adopted minor modifications 1, 2, and 3 patients (3.0%) adopted minor modifications 1, 2, and 3. All patients achieved an excellent auricle appearance and a well-laid foundation for subsequent operations. During the follow-up period, 89 patients (90.8%) were satisfied with the reconstructed auricles, 6 (6.1%) complained of hypertrophic scars in the retroauricular sulcus or pigmentation in the skin graft area, and 3 (3.1%) developed surgery-related complications. CONCLUSIONS: Three minor modifications of the auricle framework sculpting can make more satisfactory use of cartilage and adjust with the flexibility of the reconstructed auricle in different situations, making it similar to the contralateral auricle, thus, improving patients' satisfaction.

Molecular insight into the transformation of dissolved organic matter during sewage sludge composting: An investigation of a full-scale composting plant.

The aim of the study was to explore the molecular dynamics and transformation pathways of dissolved organic matter (DOM) in sewage sludge (SS) during composting, and the DOM of raw material, material experiencing thermophilic phase and material collected from humification phase were characterized using electrospray ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry. The results indicated that there were approximately 85% of aliphatic/proteins and 75% of carbohydrate preferentially decomposed in the thermophilic phase. Moreover, lignins/carboxylic-rich alicyclic molecules (CRAM) were the main N-containing substances evolved in the decomposition, which leading to a reduction of N/C ratio from 0.073 to 0.041. Whereas aliphatic acids and tryptophan in lignins/CRAM with high oxidizing capacities are preferentially decomposed in the thermophilic phase. As for maturity phase, the carbon of the newly generated compounds (belonging to lignins/CRAM and tannins), possessed an oxidation state that similar to sulfonates and sulfonamides, and these DOM are beneficial for the humic substances formation. Moreover, it was found that the newly formed N2Ox and N3Ox compounds had a more significant contribution to the double bond equivalent (DBE) of the compost, corresponding to 1.0 and 1.7 DBE, respectively. The results would help explore the understanding of DOM transformation and humification during SS composting in the microscopic molecular level.

Factors predicting recurrence after left­sided pancreatectomy for pancreatic ductal adenocarcinoma.

BACKGROUND: Recurrence after resection is the main factor for poor survival. The relationship between clinicopathological factors and recurrence after curative distal pancreatectomy for PDAC has rarely been reported separately. METHODS: Patients with PDAC after left­sided pancreatectomy between May 2015 and August 2021 were retrospectively identified. RESULTS: One hundred forty-one patients were included. Recurrence was observed in 97 patients (68.8%), while 44 (31.2%) patients had no recurrence. The median RFS was 8.8 months. The median OS was 24.9 months. Local recurrence was the predominant first detected recurrence site (n = 36, 37.1%), closely followed by liver recurrence (n = 35, 36.1%). Multiple recurrences occurred in 16 (16.5%) patients, peritoneal recurrence in 6 (6.2%) patients, and lung recurrence in 4 (4.1%) patients. High CA19-9 value after surgery, poor differentiation grade, and positive lymph nodes were found to be independently associated with recurrence. The patients receiving adjuvant chemotherapy had a decreased likelihood of recurrence. In the high CA19-9 value cohort, the median PFS and OS of the patients with or without chemotherapy were 8.0 VS. 5.7 months and 15.6 VS. 13.8 months, respectively. In the normal CA19-9 value cohort, there was no significant difference in PFS with or without chemotherapy (11.7 VS. 10.0 months, P = 0.147). However, OS was significantly longer in the patients with chemotherapy (26.4 VS. 13.8 months, P = 0.019). CONCLUSIONS: Tumor biologic characteristics, such as T stage, tumor differentiation and positive lymph nodes, affecting CA19-9 value after surgery are associated with patterns and timing of recurrence. Adjuvant chemotherapy significantly reduced recurrence and improved survival. Chemotherapy is strongly recommended in patients with high CA199 after surgery.