Enhancing alfalfa resistance to Spodoptera herbivory by sequestering microRNA396 expression.

KEY MESSAGE: Sequestering microRNA396 by overexpression of MIM396 enhanced alfalfa resistance to Spodoptera litura larvae, which may be due to increased lignin content and enhanced low-molecular weight flavonoids and glucosinolates biosynthesis. Alfalfa (Medicago sativa), the most important leguminous forage crop, suffers from the outbreak of defoliator insects, especially Spodoptera litura, resulting in heavy losses in yield and forage quality. Here, we found that the expression of alfalfa microRNA396 (miR396) precursor genes and mature miR396 was significantly up-regulated in wounding treatment that simulates feeding injury by defoliator insects. To verify the function of miR396 in alfalfa resistance to insect, we generated MIM396 transgenic alfalfa plants with significantly down-regulated miR396 expression by Agrobacterium-mediated genetic transformation. The MIM396 transgenic alfalfa plants exhibited improved resistance to Spodoptera litura larvae with increased lignin content but decreased JA accumulation. Most of the miR396 putative target GRF genes were up-regulated in MIM396 transgenic lines, and responded to the wounding treatment. By RNA sequencing analysis, we found that the differentially expressed genes related to insect resistance between WT and MIM396 transgenic plants mainly clustered in biosynthesis pathways in lignin, flavonoids and glucosinolates. In addition to the phenotype of enhanced insect resistance, MIM396 transgenic plants also displayed reduced biomass yield and forage quality. Our results broaden the function of miR396 in alfalfa and provide genetic resources for studying alfalfa insect resistance.

Brittle Culm 1 Encodes a COBRA-Like Protein Involved in Secondary Cell Wall Cellulose Biosynthesis in Sorghum.

Plant mechanical strength contributes to lodging resistance and grain yield, making it an agronomically important trait in sorghum (Sorghum bicolor). In this study, we isolated the brittle culm 1 (bc1) mutant and identified SbBC1 through map-based cloning. SbBC1, a homolog of rice OsBC1 and Arabidopsis thaliana AtCOBL4, encodes a COBRA-like protein that exhibits typical structural features of a glycosylphosphatidylinositol-anchored protein. A single-nucleotide mutation in SbBC1 led to reduced mechanical strength, decreased cellulose content, and increased lignin content without obviously altering plant morphology. Transmission electron microscopy revealed reduced cell wall thickness in sclerenchyma cells of the bc1 mutant. SbBC1 is primarily expressed in developing sclerenchyma cells and vascular bundles in sorghum. RNA-seq analysis further suggested a possible mechanism by which SbBC1 mediates cellulose biosynthesis and cell wall remodeling. Our results demonstrate that SbBC1 participates in the biosynthesis of cellulose in the secondary cell wall and affects the mechanical strength of sorghum plants, providing additional genetic evidence for the roles of COBRA-like genes in cellulose biosynthesis in grasses.

Sensitive Western-Blot Analysis of Azide-Tagged Protein Post Translational Modifications Using Thermoresponsive Polymer Self-Assembly.

Western-blot (WB) is a powerful analytical technique for protein identification in complex biological samples and has been widely used in biological studies for decades. Detection specificity and sensitivity of WB largely relies on quality of the antibodies and performance of the conjugated HRP. However, the application of WB analysis for the detection of protein post-translational modifications (PTMs) is hampered by the low abundance of protein PTMs and by the limited availability of antibodies that specifically differentiate various kinds of PTMs from their protein substrates. Therefore, new recognition mechanisms and signal amplification strategies for WB analysis of protein PTMs is in high demand. In this work, we prepared a soluble polymer that detects various azide-tagged PTM proteins in WB analysis using triarylphosphine and HRP modified thermoresponsive polymer. Specific and efficient detection of azide-tagged PTM protein is achieved via the bioorthogonal reaction between azide and triarylphosphine. More importantly, the chemiluminiscent signal in the WB analysis is largely amplified by the temperature induced self-assembly of numerous thermoresponsive polymer chains carrying multiple HRPs. As a result, approximately 100 times more sensitive detection than commercial antibodies is achieved by this method using standard PTM proteins. Though, this new reagent does not directly detect native PTMs in cell, tissue or blood samples, it still has important application potential in protein PTM studies, considering the wide availability of azide-tagging techniques to a variety of PTMs.

Preparation of polymer brushes grafted graphene oxide by atom transfer radical polymerization as a new support for trypsin immobilization and efficient proteome digestion.

Highly efficient protein digestion is one of the key issues in the "bottom-up" strategy-based proteomic studies. Compared with the time-consuming solution-based free protease digestion, immobilized protease digestion offers a promising alternative with obviously improved sample processing throughput. In this study, we proposed a new immobilized protease digestion strategy using two kinds of polymer-grafted graphene oxide (GO) conjugated trypsin. The polymer brush grafted GO was prepared using in situ polymer growth on initiator-functionalized GO using surface-initiated atom transfer radical polymerization (SI-ATRP) and characterized by AFM, TEM, TGA, and XPS. The polymer brush grafted GO supports three-dimensional trypsin immobilization, which not only increases the loading amount but also improves accessibility towards protein substrates. Both of the two types of immobilized trypsin provide 700 times shorter digestion time, while maintaining comparable protein/peptide identification scale compared with that of free trypsin digestion. More interestingly, combined application of the two types of immobilized trypsin with different surface-grafted polymers leads to at least 18.3/31.3% enhancement in protein/peptide identification compared with that obtained by digestion using a single type, indicating the potential of this digestion strategy for deeper proteome coverage using limited mass spectrometer machine hour. We expect these advantages may find valuable application in high throughput clinical proteomic studies, which often involve processing of a large number of samples. Graphical abstract Preparation of polymer brushes grafted and trypsin immobilized graphene oxide and its application in proteome digestion and mass spectrometry identification.

Preparation of sequence-controlled triblock copolymer-grafted silica microparticles by sequential-ATRP for highly efficient glycopeptides enrichment.

As one of the most important subproteomes in eukaryote cells, N-glycoproteins play crucial roles in various of biological processes and have long been considered closely correlated with the occurrence, progression, and metastasis of cancer. Comprehensive characterization of protein N-glycosylation and association of their aberrant patterns to the corresponding cancer stage may provide a unique way to discover new diagnostic biomarkers and therapeutic drug targets. However, the extremely complex nature of biological samples and relatively low abundance of N-glycosylated proteins makes the enrichment of glycoprotein/glycopeptide a prerequisite for large scale N-glycosylation identification. In this work, we prepared sequence controlled triblock copolymer grafted silica-microparticles (TCP-SMs) by sequential atom transfer radical polymerization (sequential-ATRP) of monosaccharides and zwitterionic-ion monomers for highly efficient and selective glycopeptides enrichment. The triblock copolymer is composed of sequence defined poly zwitterionic-ion, poly-N-acetylglucosamine and poly mannose blocks. The glycopolymer blocks carrying densely packed pendent sugars are excellent mimics of the natural carbohydrate clusters and may induce multivalent carbohydrate-carbohydrate interaction (CCI) with the target glycopeptides. Therefore, increased retention of glycopeptides can be expected by the combination of CCI and zwitterionic-HILIC interaction. As a result, 1244 glycopeptides were identified after TCP-SMs enrichment from mouse liver, which are 65-120% higher than that obtained by homoglycopolymer or random-copolymer grafted silica microparticles prepared using the conventional free radical polymerization. These results demonstrate the critical role of sequence-defined block copolymer of TCP-SMs for obtaining enhanced affinity toward glycopeptides and the potential of this sequential-ATRP strategy to integrate different affinity moieties into one enrichment material to achieve deeper coverage in protein PTM mapping.

Enhanced Agrobacterium-mediated transformation efficiencies in monocot cells is associated with attenuated defense responses.

Plant defense responses can lead to altered metabolism and even cell death at the sites of Agrobacterium infection, and thus lower transformation frequencies. In this report, we demonstrate that the utilization of culture conditions associated with an attenuation of defense responses in monocot plant cells led to highly improved Agrobacterium-mediated transformation efficiencies in perennial ryegrass (Lolium perenne L.). The removal of myo-inositol from the callus culture media in combination with a cold shock pretreatment and the addition of L-Gln prior to and during Agrobacterium-infection resulted in about 84 % of the treated calluses being stably transformed. The omission of myo-inositol from the callus culture media was associated with the failure of certain pathogenesis related genes to be induced after Agrobacterium infection. The addition of a cold shock and supplemental Gln appeared to have synergistic effects on infection and transformation efficiencies. Nearly 60 % of the stably transformed calluses regenerated into green plantlets. Calluses cultured on media lacking myo-inositol also displayed profound physiological and biochemical changes compared to ones cultured on standard growth media, such as reduced lignin within the cell walls, increased starch and inositol hexaphosphate accumulation, enhanced Agrobacterium binding to the cell surface, and less H(2)O(2) production after Agrobacterium infection. Furthermore, the cold treatment greatly reduced callus browning after infection. The simple modifications described in this report may have broad application for improving genetic transformation of recalcitrant monocot species.

Trypsin immobilization on hairy polymer chains hybrid magnetic nanoparticles for ultra fast, highly efficient proteome digestion, facile 18O labeling and absolute protein quantification.

In recent years, quantitative proteomic research attracts great attention because of the urgent needs in biological and clinical research, such as biomarker discovery and verification. Currently, mass spectrometry (MS) based bottom up strategy has become the method of choice for proteomic quantification. In this strategy, the amount of proteins is determined by quantifying the corresponding proteolytic peptides of the proteins, therefore highly efficient and complete protein digestion is crucial for achieving accurate quantification results. However, the digestion efficiency and completeness obtained using conventional free protease digestion is not satisfactory for highly complex proteomic samples. In this work, we developed a new type of immobilized trypsin using hairy noncross-linked polymer chains hybrid magnetic nanoparticle as the matrix aiming at ultra fast, highly efficient proteomic digestion and facile (18)O labeling for absolution protein quantification. The hybrid nanoparticle is synthesized by in situ growth of hairy polymer chains from the magnetic nanoparticle surface using surface initiated atom transfer radical polymerization technique. The flexible noncross-linked polymer chains not only provide large amount of binding sites but also work as scaffolds to support three-dimensional trypsin immobilization which leads to increased loading amount and improved accessibility of the immobilized trypsin. For complex proteomic samples, obviously increased digestion efficiency and completeness was demonstrated by 27.2% and 40.8% increase in the number of identified proteins and peptides as well as remarkably reduced undigested proteins residues compared with that obtained using conventional free trypsin digestion. The successful application in absolute protein quantification of enolase from Thermoanaerobacter tengcongensis protein extracts using (18)O labeling and MRM strategy further demonstrated the potential of this hybrid nanoparticle immobilized trypsin for high throughput proteome quantification.

Robust Dual-Biomimetic Titanium Dioxide-Cellulose Monolith for Enrichment of Phosphopeptides.

Metal oxide affinity chromatography (MOAC) is considered to be one of the most effective methods for phosphopeptide enrichment. However, most of the materials used in the method are powder; frequent centrifugation is necessitated during the enrichment process, and potential risks of loss of peptides and materials and clogging of the column employed for liquid chromatography-mass spectrometry (LC-MS) arise. Moreover, the reusability of these materials to achieve sustainability was hardly investigated. To overcome these limitations, herein, inorganic titanium dioxide (TiO2) was coated onto the skeletal surface of the organic cellulose monolith (CM) material with a coral-like structure via a sol-gel method. This produced an organic-inorganic hybrid TiO2-CM material, which contained a combination of organic and inorganic substances, making it mimic the mollusk shell in terms of composition. The prepared TiO2-CM material as monolith exhibited excellent mechanical strength and did not break during the enrichment process; thus, the tedious implementation of multiple centrifugation cycles was prevented, thereby streamlining the experimental procedure and avoiding the loss of peptides and materials. Moreover, a large amount of TiO2 was introduced onto the surface of the CM material, and thus, the resultant TiO2-CM material exhibited a large surface area. As a result, the fabricated TiO2-CM material was successfully applied to the enrichment of phosphopeptides obtained from the tryptic digests of a BSA/ß-casein (mass ratio, 500/1) mixture. The results were superior to those achieved for commercial TiO2 beads, confirming that TiO2-CM has excellent selectivity for phosphopeptides and reusability. Furthermore, 9287 unique phosphopeptides derived from the 2661 phosphoproteins were successfully identified from two milligrams of tryptic digests of Hela cell exosomes obtained through five independent replications after enriching using the TiO2-CM material. The results indicated that the material has good application prospects in the analysis of protein phosphorylation. Furthermore, TiO2-CM consists of green and cheap cellulose as the skeleton, and its synthesis process is environment-friendly, simple, and inexpensive.

Optimizing anaerobic technology by using electrochemistry and membrane module for treating pesticide wastewater: Chemical oxygen demand components and fractions distribution, membrane fouling, effluent toxicity and economic analysis.

Optimization in performance and membrane fouling of an electrochemical anaerobic membrane bioreactor (R1) for treating pesticide wastewater was investigated and compared with a conventional anaerobic membrane bioreactor (R2). The maximum COD removal efficiency of R2 was 80.1%, 80.0%, 67.4%, 61.1% with HRT of 96, 72, 48 and 24 h, which of R1 was enhanced to 84.7%, 84.3%, 82.0% and 66.3%. These results demonstrated that the optimum HRT of R1 was shortened to 48 h, which of R2 required 72 h. R1 reduced the contents of particulate and colloidal COD, and the fraction of COD converted to sludge was 5.0-8.2% lower than that of R2. The fouling rate was 0.99-1.44 kPa/d and reduced by 31.0%-38.5% compared with R2. Detoxification was enhanced by 7.8-47.7% with the assistance of bio-electrochemistry. Ultimately, ensuring similar performance, R1 achieved a 65.6% improvement in environmental benefit, a 26.3% and 38.9% reduction in unit capital and operating costs.

The clinical study of CBCT imaging technology in the restoration of upper anterior teeth of the elderly.

OBJECTIVE: To evaluate and analyze the clinical effect of CBCT imaging technology on the restoration of upper anterior teeth of the elderly. METHODS: 36 elderly patients with upper anterior teeth loss in our hospital from January 2018 to January 2020 were selected for implant restoration. Patients were equally randomized into a curved tomographic restoration group (TR group) and a CBCT restoration group (CR group). Patients in the two groups underwent traditional implant restoration. Then we compared and analyzed the implant migration, the adjustment time of first wearing, and the success rate of axial gingival recession and restoration satisfaction of patients in the two groups. RESULTS: The neck offset and the root offset of the implants in the CR group was (0.77±0.15) mm and (0.83±0.17) mm, respectively, which were significantly lower than (1.25±0.27) mm and (1.73±0.29) mm in the TR group (t=6.593, t=11.359, all P<0.01). The initial wearing adjustment time of patients in the CR group [(8.73±1.94) min] was significantly less than (18.79±4.85) min in the TR group (t=8.171, P<0.01); the CR group had a significantly higher success rate of axial gingival recession as compared to the CR group (94.44% vs 61.11%, χ2=6.0857, P<0.05); The restoration satisfaction rate of patients in the CR group was 100%, which was significantly higher than 77.78% of the TR group (χ2=8.7429, P<0.05). CONCLUSION: The CBCT imaging technology has a significant clinical effect on the restoration of the upper anterior teeth of the elderly, which effectively reduces the deviation of implant placement, shorten the adjustment time of their initial wearing, and greatly improves the success rate of axial gingival recession, effectively guarantees the long-term stability and aesthetics of dental implant restoration, and significantly enhances the satisfaction of patients.

A global review of rubber plantations: Impacts on ecosystem functions, mitigations, future directions, and policies for sustainable cultivation.

The growing global need for latex is driving rubber plantation (RP) expansion since the last century, with >2 Mha of cultivation area being established in the last decade. Southeast Asia is the hotspot for rubber cultivation at other land-use costs. Although rubber cultivation has improved the economic status of farmers, it has altered the habitat's ecology and ecosystem functions (EF). However, studies on the impacts of RP on EF are limited, and a clear overview is not available. To bridge this gap, we conducted an inclusive review of the EF of RP, including soil carbon storage, aboveground biomass (AGB) and belowground biomass (BGB), litter production and decomposition, respiration, and biodiversity (plants, animals, soil fauna, and microbes). We compared the EF in RP (monoculture) with those in forests because the conversion of forests to RP is prevalent in the tropics and because most RP studies used forests as reference ecosystems. We found RP generally have lower EF than forests. The impacts of RP on some EF are more severe (e.g., AGB, BGB, and plant diversity), causing decreases of >55%, and the effects are consistently negative irrespective of plantation age. However, including agroforestry or polyculture, integrated pest management, cover cropping, mulching, and composting can improve the EF in RP to some extent. We highlighted research gaps, particularly substantial research gaps concerning the influence of plant diversity treatments (i.e., agroforestry) performed in RP on EF. Additionally, more empirical data on the significance of spatial and temporal levels are required, such as how the impact on EF could vary with climate and RP age, as we showed some examples where EF differs spatially and temporally. More importantly, further research on plantation management to offset EF losses is needed. Finally, we emphasized knowledge gaps and suggested future directions and policies for improving EF in RP.

Surface initiated atom transfer radical polymerization: access to three dimensional wavelike polymer structure modified capillary columns for online phosphopeptide enrichment.

Reversible phosphorylation is one of the most important post-translational modifications of proteins and a key regulator of cellular signaling pathways. Specific enrichment of phosphopeptides from proteolytic digests is a prerequisite for large scale identification of protein phosphorylation by mass spectrometry. Online enrichment of phosphopeptides attracts particular interests due to its automated operation, higher throughput and reproducibility, lower sample loss, and contamination. Here, we report a new type of capillary column developed using surface initiated atom transfer radical polymerization (SI-ATRP) for automated online phosphopeptide enrichment. SI-ATRP modification leads to a surface confined growth of three-dimensional wavelike polymer structure on the inner wall of capillary columns and, therefore, results in largely increased surface area. Furthermore, the noncross-linked flexible polymer chains grown by SI-ATRP create a large internal volume that allows phosphopeptides to penetrate into during enrichment and also facilitate the interaction between the numerous functional groups in the polymer chains and target phosphopeptides. Therefore, highly efficient and specific enrichment is achieved even for a low femtomole of phosphopeptides. The loading capacity is increased more than an order of magnitude compared with that obtained using conventional open tubular capillary columns. The SI-ATRP modified capillary column was successful applied in the online phosphoproteomics analysis of HepG2 cell lysate and resulted in 10 times improved phosphopeptide identification than the previously reported number. Finally, the SI-ATRP technique is compatible with a variety of functional monomers, and therefore, versatile potential applications in reverse phase, ion exchange, and affinity chromatography can be expected.

Interferon and lamivudine combination therapy versus lamivudine monotherapy for hepatitis B e antigen-negative hepatitis B treatment: a meta-analysis of randomized controlled trials.

BACKGROUND: It has been demonstrated that only a minority of patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB) obtain a sustained response after either interferon (IFN) or nucleos(t)ide analogue monotherapy. Therefore, combination therapy of drugs with synergistic antiviral effects was proposed to have a sustained response in these patients. We compared the effect and safety of lamivudine monotherapy and its combination with IFN including conventional interferon (CON-IFN) and pegylated interferon (PEG-IFN) for HBeAg-negative CHB patients. DATA SOURCES: A group of three independent reviewers identified 9 eligible randomized controlled trials through electronic searches (MEDLINE, OVID, EMBASE, the Cochrane Library Clinical Trials Registry, and the Chinese Medical Database), manual searches, and contact with experts. Sustained virological and biochemical responses were defined as primary efficacy measures. We performed quantitative meta-analyses to assess differences between CON-IFN plus lamivudine combination and lamivudine monotherapy groups. RESULTS: No greater sustained virological and biochemical rates were found in patients receiving CON-IFN/lamivudine combination therapy [29.1% vs. 26.7%, odds ratio (OR)=0.98, 95% confidence interval (CI) 0.65-1.50, P=0.94, and 41.8% vs. 40.3%, OR=1.13, 95% CI 0.78-1.65, P=0.51, respectively], though a reduced YMDD mutation rate was achieved in the combination group [8.39% vs. 30.0%, OR=0.16, 95% CI 0.076-0.33, P<0.001]. However, data from one PEG-IFN trial showed greater sustained virological and biochemical rates in patients receiving combination therapy [response rate 19.5% vs. 6.6%, OR=3.42, 95% CI 1.71-6.84, P<0.001 and 60.0% vs. 44.2%, OR=1.88, 95% CI 1.23-2.85, P=0.003, respectively]. CONCLUSIONS: Addition of CON-IFN to lamivudine did not improve treatment efficacy but suppressed YMDD mutation by lamivudine. Combination of PEG-IFN and lamivudine might increase the sustained response, and further clinical trials are needed for confirmation.

Recognizing the role of plant species composition in the modification of soil nutrients and water in rubber agroforestry systems.

Reliable guidance for crop selection and related management to achieve sustainable soil resource use in rubber agroforestry systems is limited. One important reason for this limited guidance is that our understanding of the effects of different plant functional groups on soil resources is still insufficient. Here, to examine the effects of the species composition of trees, shrubs and herbs on soil nutrients and soil water with increases in the complexity of the plant community structure, we measured the soil nutrient concentrations (i.e., C, N, P, K, Ca and Mg), soil water content and soil water residence time (with stable hydrogen and oxygen isotope tracers) at six soil depths in a monoculture rubber plantation, four multi-species rubber agroforestry systems, and a tropical rainforest. As the plant species composition increased, the soil C and N increased. The soil water content also increased with increases in soil C and N. However, the effects of plant species composition on the soil water content gradually changed from positive to negative, especially under the effects of herb species, which could accelerate soil water drainage and hence shorten the soil water residence time. Therefore, the faster water infiltration and potentially higher flow of soil water in complex plant communities increased the risk and magnitude of mineral nutrient leaching. In addition, as the plant composition increased, plant competition decreased the concentration of soil nutrients, especially soil P, K and Ca. In general, plant interspecific interactions definitively decreased soil mineral nutrients as the plant composition increased, and the effects of tree, shrub and herb species on soil nutrients and soil water differed and sometimes appeared contradictory. However, the effects of plant species composition on soil gradually weakened with increases in soil depth.

PvNAC1 increases biomass and enhances salt tolerance by decreasing Na+ accumulation and promoting ROS scavenging in switchgrass (Panicum virgatum L.).

Switchgrass (Panicum virgatum L.) is a bioenergy crop; thus, it is important to improve biomass to effectively produce bioethanol, particularly under adverse stress conditions. NAC transcription factors are involved in the abiotic stress response. PvNAC1 was isolated in the nucleus of switchgrass, with its C-terminal region containing a transcriptional activation domain. PvNAC1 expression was induced by dehydration, salt, H2O2, and abscisic acid treatments. Overexpressing (OE) PvNAC1 improved growth performance, leading to significantly taller and heavier (dry weight) plants. Moreover, cellulose content was significantly higher in OE plants, indicating that PvNAC1 plays an important role regulating growth and bioethanol production. PvNAC1 RNA interference (RNAi) switchgrass plants exhibited reduced dry weight and cellulose content. OE PvNAC1 enhanced tolerance to salt stress, through higher reactive oxygen species scavenging ability and less Na+ and more K+ accumulation in roots and shoots. RNAi plants were more sensitive to salt stress. The quantitative polymerase chain reaction results revealed that some stress responsive genes, three antioxidant enzymatic genes, and an ion homeostasis-related gene were upregulated in OE plants and downregulated in RNAi plants. These results show that PvNAC1 functions as a transcriptional activator in response to salt stress and growth.

Multiple primary malignant neoplasms of three early cancer lesions: a case report.

Multiple primary malignant neoplasms (MPMNs) are rarely reported and it is important to give early diagnosis and proper therapy for these patients. Here reported a case of 62-year-old man with concomitant three early stage cancer lesions in upper gastrointestinal tract, all of which were detected by endoscopy. The first one was an IIc-type lesion at angular part of stomach under endoscopy, which was histologically confirmed to be a mucosal well-differentiated adenocarcinoma. The patient underwent a standard radical gastrectomy for the lesion after the failure of endoscopic treatment. The other two neoplasms were observed during follow-up and were indicated as early stage lesions by synthesizing information from endoscopy, endoscopic ultrasonography, computed tomography and biopsy. One displayed as a hyperemic patch (3 cm×4 cm in size) located at the part of esophagus 27 cm away from the incisor teeth and was proved to be moderately differentiated squamous cancer by histopathological examination. The other was an IIc-type lesion (3.0 cm×3.5 cm in size) located at the part of esophagus 36 cm away from the incisor teeth, and the biopsy result showed a poorly differentiated squamous carcinoma. Both the two lesions were treated with radical radiation because the patient refused surgery management. No recurrence of former lesions or occurrence of novel lesions were observed during post-treatment follow-up, suggesting radical radiation might be effective for this patient.