Micro-/nanostructures for surface-enhanced Raman spectroscopy: Recent advances and perspectives.

Surface-enhanced Raman spectroscopy (SERS) has great potential for the analysis of molecules adsorbed on metals with rough surfaces or substrates with micro-/nanostructures. Plasmonic coupling between metal nanoparticles and the morphology of the rough metal surface can produce "hot spots" that enhance Raman scattering by adsorbed molecules, typically at micro- to nanomolar concentrations, although high enhancement factors can also facilitate single-molecule detection. This phenomenon is widely applicable for chemical analysis and sensing in various fields. In this review, the latest research progress on SERS micro-/nanosensors is evaluated, and the sensors are classified according to their individual functions. Furthermore, the design principles and working mechanisms of reported SERS-active micro-/nanostructured substrates are analyzed, and the design features adopted to overcome the difficulties associated with precision detection are explored. Finally, challenges and directions for future development in this field are discussed. This review serves as a design guide for novel SERS-active substrates.

Synthetic biology for the food industry: advances and challenges.

As global environmental pollution increases, climate change worsens, and population growth continues, the challenges of securing a safe, nutritious, and sustainable food supply have become enormous. This has led to new requirements for future food supply methods and functions. The use of synthetic biology technology to create cell factories suitable for food industry production and renewable raw material conversion into: important food components, functional food additives, and nutritional chemicals, represents an important method of solving the problems faced by the food industry. Here, we review the recent progress and applications of synthetic biology in the food industry, including alternatives to: traditional (artificial pigments, meat, starch, and milk), functional (sweeteners, sugar substitutes, nutrients, flavoring agents), and green (green fiber, degradable packing materials, green packaging materials and food traceability) foods. Furthermore, we discuss the future prospects of synthetic biology-based applications in the food industry. Thus, this review may serve as a reference for research on synthetic biology in the: food safety, food nutrition, public health, and health-related fields.

A comparative study of INSM1 (clone MRQ70) immunoreactivity on CytoLyt® pretreated cytology and resection specimens of pancreatic neuroendocrine tumors.

INTRODUCTION: Insulinoma-associated protein 1 (INSM1) is a newly characterized sensitive and specific immunohistochemical marker for neuroendocrine (NE) tumors. Whereas more traditional NE markers, such as chromogranin A and synaptophysin, are cytoplasmic, INSM1 is uniquely nuclear and thus could serve as a useful addition to NE tumor workup. While application of immunohistochemical studies to cytology specimens is becoming increasingly relevant, knowledge of the effects of the certain fixatives as well as the pattern and intensity of immunoexpression are important considerations. METHODS: Sixteen cases of pancreatic neuroendocrine tumor (PanNET) diagnosed between 2015 and 2021 underwent both fine-needle aspiration, which was subsequently prepared in CytoLyt®-fixed cytology cell block (CCB), and surgical resection, in which specimens were prepared into formalin-fixed paraffin embedded blocks (FFPE). For all samples, INSM1 immunoreactivity was classified according to staining intensity and extent, then compared between CCBs and matched FFPEs. RESULTS: All 16 FFPE specimens demonstrated strong and diffuse INSM1 immunoreactivity, while only 10/16 (62.5%) CCBs were positive. Of those 10, only 2/10 (20%) demonstrated strong and diffuse reactivity. CONCLUSION: The choice of fixative has a demonstrable effect on the immunoreactivity of INSM1 in PanNET. Even though the sensitivity is lower in CytoLyt®-fixed cell block specimens, the addition of INSM1 is useful, especially in challenging cases that may be negative for one or more of the traditional NE markers.

Carboxyl-functionalized two-dimensional MXene-Au nanocomposites were prepared as SERS substrates for the detection of melamine in dairy products.

In the present study, we address the limitations of conventional surface-enhanced Raman scattering (SERS) techniques for sensitive and stable detection of melamine in food products, especially dairy. To overcome these challenges, we developed a novel SERS-active substrate by incorporating gold nanoparticles (AuNPs) onto carboxyl-functionalized two-dimensional (2D) MXene material doped with nitrides, specifically Au-Ti2N-COOH. Our strategy leverages the unique physicochemical properties of MXene, a class of atomically thin, 2D transition metal carbides/nitrides, with tunable surface functionalities. By modifying the MXene surface with AuNPs and introducing carboxyl groups (-COOH), we successfully enhanced the interaction between the substrate and melamine molecules. The carboxyl groups form hydrogen bonds with the amino groups on the melamine's triazine ring, facilitating the adsorption of melamine molecules within the 'hotspot' regions responsible for SERS signal amplification. A series of characterization methods were used to confirm the successful synthesis of Au-Ti2N-COOH composites.Using Au-Ti2N-COOH as the SERS substrate, we detected melamine in spiked dairy product samples with significantly enhanced sensitivity and stability compared to nitride-doped MXene alone. The detection limit in liquid milk stands at 3.7008 µg kg-1, with spike recovery rates ranging from 99.84% to 107.55% and an approximate RSD of 5%. This work demonstrates the effectiveness of our approach in designing a label-free, rapid, and robust SERS platform for the accurate quantitation of melamine contamination in food, thereby mitigating health risks associated with melamine adulteration.

Rapid detection of melamine by DNA Walker mediated SERS sensing technique based on signal amplification function.

A new method is proposed for detecting typical melamine dopants in food using surface-enhanced Raman scattering (SERS) biosensing technology. Melamine specific aptamer was used as the identification probe, and gold magnets (AuNPs@MNPs) and small gold nanoparticles (AuNPs@MBA) were used as the basis for Raman detection. The Raman signal of the detection system can directly detect melamine quantitatively. Under optimized conditions, the detection of melamine was carried out in the low concentration range of 0.001-500 mg/kg, the enhancement factor (EF) was 2.3 × 107, and the detection limit was 0.001 mg/kg. The method is sensitive and rapid, and can be used for the rapid detection of melamine in the field environment.

Multisignal Biosensors Based on Mn Paramagnetic Relaxation and Nanocatalysis for Norovirus Detection.

A multisignal method for the sensitive detection of norovirus based on Mn paramagnetic relaxation and nanocatalysis was developed. This dual-modality sensing platform was based on the strong relaxation generated by cracked Au@MnO2 nanoparticles (NPs) and their intrinsic enzyme-like activity. Ascorbic acid rapidly cracked the MnO2 layer of Au@MnO2 NPs to release Mn(II), resulting in the relaxation modality being in a "switch-on" state. Under the optimal conditions, the relaxation modality exhibited a wide working range (6.02 × 103-3.01 × 107 copies/µL) and a limit of detection (LOD) of 2.29 × 103 copies/µL. Using 4,4',4″,4″'-(porphine-5,10,15,20-tetrayl) tetrakis (benzenesulfonic acid) (tpps)-ß-cyclodextrin (tpps-ß-CD) as a T1 relaxation signal amplification reagent, a lower LOD was obtained. The colorimetric modality exploited the "peroxidase/oxidase-like" activity of Au@MnO2 NPs, which catalyzed the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue oxidized TMB, which exhibited a working range (6.02 × 104-6.02 × 106 copies/µL) and an LOD of 2.6 × 104 copies/µL. In addition, the rapid amplification reaction of recombinase polymerase enabled the detection of low norovirus levels in food samples and obtained a working range of 101-106 copies/mL and LOD of 101 copies/mL (relaxation modality). The accuracy of the sensor in the analysis of spiked samples was consistent with that of the real-time quantitative reverse transcription polymerase chain reaction, demonstrating the high accuracy and practical utility of the sensor.

Programmable DNA tweezers-SDA for ultra-sensitive signal amplification fluorescence sensing strategy.

BACKGROUND: DNA tweezers, classified as DNA nanomachines, have gained prominence as multifunctional biosensors due to their advantages, including a straightforward structure, response mechanism, and high programmability. While the DNA tweezers demonstrate simultaneous, rapid, and stable responses to different targets, their detection sensitivity requires enhancement. Some small molecules, such as mycotoxins, often require more sensitive detection due to their extremely high toxicity. Therefore, more effective signal amplification strategies are needed to further enhance the sensitivity of DNA tweezers in biosensing. RESULTS: We designed programmable DNA tweezers that detect small-molecule mycotoxins and miRNAs through simple sequence substitution. While the DNA tweezers demonstrate simultaneous, rapid, and stable responses to different targets, their detection sensitivity requires enhancement. We introduced the Strand Displacement Amplification (SDA) technique to address this limitation, proposing a strategy of novel programmable DNA tweezers-SDA ultrasensitive signal amplification fluorescence sensing. We specifically investigate the effectiveness of this approach concerning signal amplification for two critical mycotoxins: aflatoxin B1 (AFB1) and zearalenone (ZEN). Results indicate that the detection ranges of AFB1 and ZEN via this strategy were 1-10,000 pg mL -1 and 10-100,000 pg mL -1, respectively, with corresponding detection limits of 0.933 pg mL -1 and 1.07 pg mL -1. Compared with the DNA tweezers direct detection method for mycotoxins, the newly constructed programmable DNA tweezers-SDA fluorescence sensing strategy achieved a remarkable 104-fold increase in the detection sensitivity for AFB1 and ZEN. SIGNIFICANCE: The constructed programmable DNA tweezers-SDA ultrasensitive signal-amplified fluorescence sensing strategy exhibits excellent detection performance for mycotoxins. The superb versatility of this strategy allows the developed method to be easily used for detecting other analytes by simply replacing the aptamer and cDNA, which has incredible potential in various fields such as food safety screening, clinical diagnostics, and environmental analysis.

Machine learning assisted biosensing technology: An emerging powerful tool for improving the intelligence of food safety detection.

Recently, the application of biosensors in food safety assessment has gained considerable research attention. Nevertheless, the evaluation of biosensors' sensitivity, accuracy, and efficiency is still ongoing. The advent of machine learning has enhanced the application of biosensors in food security assessment, yielding improved results. Machine learning has been preliminarily applied in combination with different biosensors in food safety assessment, with positive results. This review offers a comprehensive summary of the diverse machine learning methods employed in biosensors for food safety. Initially, the primary machine learning methods were outlined, and the integrated application of biosensors and machine learning in food safety was thoroughly examined. Lastly, the challenges and limitations of machine learning and biosensors in the realm of food safety were underscored, and potential solutions were explored. The review's findings demonstrated that algorithms grounded in machine learning can aid in the early detection of food safety issues. Furthermore, preliminary research suggests that biosensors could be optimized through machine learning for real-time, multifaceted analyses of food safety variables and their interactions. The potential of machine learning and biosensors in real-time monitoring of food quality has been discussed.

Application of DNA-fueled molecular machines in food safety testing.

Food is consumed by humans, which is indispensable to human life. Therefore, considerable attention of the whole society has been paid to food safety. Over the last few years, dramatic social development has brought new challenges to food safety, making developing new and quick methods for on-site food safety testing an important necessity. As a result, DNA-fueled molecular machines, characterized by high efficiency, accuracy, and sensitivity in testing, have come into the spotlight, based on which sensors can be constructed to detect toxic and harmful substances in food products. This study reviewed recent research on several DNA-fueled molecular machines, including DNA tweezers, DNA walkers, and DNA origami, for rapidly detecting toxic and harmful substances. Based on the above studies, the sensitivity and timeliness of several DNA molecular machines were summarized and compared, and the development prospect of DNA fuel molecular machines in the field of food safety detection was prospected.

MOF-on-MOF heterostructure boosting AIE sensing and triggered structural collapse for histamine detection.

We explored a novel preparation method for MOF-on-MOF heterostructured material (Zn-BTEC@ZIF-8). This prepared heterostructured material acts as a container, capable of adsorbing tetracycline hydrochloride molecules into its backbone through hydrogen bonding and π-π interactions. This phenomenon triggers an aggregation induced emission (AIE) effect, leading to the formation of luminescent bodies. The coordination between histamine and MOF was found to collapse the originally stabilized MOF-on-MOF structure. This collapse causes the splitting of the initially stabilized MOF-on-MOF structure from the aggregated state into fragments, resulting in the quenching of fluorescence in the fluorophore. Remarkably, the fluorescence quenching efficiency of this composite surpasses that of single-layer metal-organic framework (MOF) zeolitic imidazolate framework-8 (ZIF-8) or zinc-based MOF of pyromellitic acid (Zn-BTEC), enabling more sensitive detection of histamine. In this investigation, we constructed a label-free fluorescent sensor specifically designed for the detection of histamine, capitalizing on the AIE effect inherent in MOF-on-MOF architecture and the presence of tetracycline hydrochloride (Tet). The sensor demonstrates a rapid, straightforward, and stable response, allowing for histamine detection within 20 min. Notably, the sensor covers a detection range of 2-400 mg L-1, achieving a low detection limit of 1.458 mg L-1 The practical application of this sensor for quantitative detection of histamine in river water and various fish species exhibited robust performance, ensuring reliability and accuracy in real samples. Its potential application in food safety and environmental monitoring is evident, making it a valuable tool for addressing histamine-related challenges in these domains.

Ni-Pt nanozyme-mediated relaxation and colorimetric sensor for dual-modality detection of norovirus.

An NiPt nanozyme-mediated relaxation and colorimetric sensor is developed for dual-modality detection of norovirus (NoV). The relaxation modality is based on the "catalase-like" activity of the NiPt nanozyme, which adjusts the hydrogen peroxide (H2O2) mediated Fe (II)/Fe(III) conversion, thereby changing the relaxation signal. Poly-γ-glutamic acid (MW ≈ 1w) can enhance the relaxivity of Fe(III) (r1 = 7.11 mM-1 s-1; r2 = 8.94 mM-1 s-1). The colorimetric modality exploits the "peroxidase-like" activity of the NiPt nanozyme, which can catalyze the oxidation of colorless 3, 3', 5, 5'-tetramethylbenzidine (TMB) to blue oxTMB in H2O2. Under optimal conditions, the relaxation modality exhibits a wide working range (1.0 × 101-1.0 × 104 fM) and a limit of detection (LOD) of 4.7 fM (equivalent to 2820 copies/µL). The spiked recoveries range from 99.593 to 106.442 %, and the relative standard deviation (RSD) is less than 5.124 %. The colorimetric modality exhibited the same working range with a lower LOD of 2.9 fM (equivalent to 1740 copies/µL) and an RSD of less than 2.611 %. Additionally, the recombinase polymerase amplification reaction enabled the detection of low NoV levels in food samples with a working range of 102-106 copies/mL and LOD of 102 copies/mL. The accuracy of the sensor in the analysis of spiked samples is consistent with the gold standard method (real-time quantitative reverse transcription-polymerase chain reaction), demonstrating the high accuracy and practical utility of the sensor.

A facile dual-mode immunosensor based on speckle Ag-doped nanohybrids for ultrasensitive detection of Ochratoxin A.

Ochratoxin A (OTA) is a potent carcinogen, and is among the most dangerous mycotoxins in agricultural products. In this study, an ultrasensitive dual-mode immunosensor was developed for naked-eye and fluorescence detection of OTA based on Ag-doped core-shell nanohybrids (Ag@CSNH). Complete antigen-labeled Ag@CSNH (CA-Ag@CSNH) were used as a competitive bind and dual-mode probe. The diffused doping structure of CA-Ag@CSNH provided improved stability, color and fluorescence quencher performance. Antibodies modified magnetic beads were used as a capture probe. The competitive binding between OTA and CA-Ag@CSNH produced both color change and fluorescence quenching. Ultraviolet and fluorescence intensitie correlated linearly with OTA concentration ranges of 0.03-3 ng/mL and 10-10000 pg/mL, and limits of detection of 0.0235 ng/mL and 0.9921 pg/mL, respectively. The practical applicability of proposed strategy was demonstrated by analysis of OTA in spiked corn, soybean and flour samples. This study offers a new insight on multi-mode platforms for various applications.

Classification and Quantitative Characterization of Archean Metamorphic Buried Hill Reservoirs in the Bohai Sea.

Based on productivity test data and physical property test results from multiple wells, a classification scheme of Archean metamorphic buried hill reservoirs in the Bohai Sea is established by means of mathematical function fitting. By combining data from cores, casting thin sections, scanning electron microscopy, imaging logging, and high-pressure mercury injection and nitrogen adsorption tests, we clarified the reservoir composition and pore structure characteristics of different types of reservoirs are clarified. Furthermore, taking the BZ19-6 and 13-2 wells in the Archean metamorphic buried hills as an example, the development sites of different types of reservoirs are analyzed and the reservoir development model is established. The results show that the Archean metamorphic buried hill reservoirs in the Bohai Sea can be divided into three categories and six subcategories, including type I reservoirs with porosities greater than 8% or permeabilities greater than 1 × 10-3 µm2 and type II reservoirs with porosities of 5-8% or permeabilities in the range of 0.1-1 × 10-3 µm2. Reservoirs with porosities of 2-5% and permeabilities of 0.01-0.1 × 10-3 µm2 are type III reservoirs. Each type of reservoir can be further divided into a fracture-pore type and a fracture type according to the relative contribution of the porosity and permeability to the reservoir. From type I to type III, the dissolution degree and fracture development gradually weaken, the pore size gradually decreases, and the pore volume gradually decreases. The distribution of favorable reservoirs is comprehensively controlled by weathering and tectonic transformation. The presence of a weathered glutenite zone, weathered leaching zone, or weathered disintegration zone is favorable for the development of type I reservoirs in the weathering crust. In the inner part of the buried hill, the presence of a fracture zone with a thickness of more than 10 m or a dense fracture zone with a thickness of more than 40 m is favorable for the formation of type I reservoirs.

A multifunctional polyacrylamide/chitosan hydrogel for dyes adsorption and metal ions detection in water.

Removing noxious dyes and detecting excessive metal ions in water are both effective means to prevent damage from contaminants and ensure water safety. The emphasis problems were addressed by preparation a polyacrylamide chitosan (PAAM/CS) hydrogel. Polyacrylamide (PAAM) provides overall mechanical strength to carry loads and facilitate circulation, chitosan (CS) provides adsorption positions with high adsorption capacity. Which made that PAMM/CS hydrogel efficiently performed sorption of xylenol orange (XO). As the functional dye, XO binds to PAAM/CS and confers colorimetric properties on PAAM/CS hydrogels. XO sorbed hydrogel realized fluorescence dual-signal detection of Fe3+ and Al3+ in water. The significant swelling and adsorption potency of the hydrogel, combined with the dual-signal detection capability of XO sorbed hydrogel, make this hydrogel a versatile material for environmental applications.

Application of DNA Nanotweezers in biosensing: Nanoarchitectonics and advanced challenges.

Deoxyribonucleic acid (DNA) is a carrier of genetic information. DNA hybridization is characterized by predictability, diversity, and specificity owing to the strict complementary base-pairing assembly mode, which stimulates the use of DNA to build a variety of nanomachines, including DNA tweezers, motors, walkers, and robots. DNA nanomachines have become prevalent for signal amplification and transformation in the field of biosensing, providing a new method for constructing highly sensitive sensing analysis strategies. DNA tweezers have exhibited unique advantages in biosensing applications owing to their simple structures and fast responses. The two-state conformation of DNA tweezers, the open and closed states, enable them to open and close autonomously after stimulation, thus facilitating the quick detection of corresponding signal changes of different targets. This review discusses the recent progress in the application of DNA nanotweezers in the field of biosensing, and the trends in their development for application in the field of biosensing are summarized.

Functionalized magnetic nanobeads for SERS-based detection of Staphylococcus aureus.

In this study, we introduced a Raman detection technique based on a combination of functionalized magnetic beads and surface-enhanced Raman scattering (SERS) tags to develop a rapid and sensitive strategy for the detection of Staphylococcus aureus (S. aureus), a typical foodborne pathogen. Polyethylene glycol (PEG) and bovine serum albumin (BSA) dual-mediated teicoplanin functionalized magnetic beads (TEI-BPBs) were prepared for separation of target bacteria. SERS tags were used to immobilize antibodies on gold surfaces with bifunctional linker proteins to ensure specific recognition of S. aureus. Under optimal conditions, the combination of TEI-BPBs and SERS tags showed reliable performance, exhibiting good capture efficiency even in the presence of 106 CFU mL-1 of non-target bacteria. The SERS tag provided an effective hot spot for subsequent Raman detection, presenting good linearity in the range of 102-107 CFU mL-1. Good performance has also been shown in detecting target bacteria in milk samples, where it has a recovery of 95.5-101.3%. Thus, the highly sensitive Raman detection technique combined with TEI-BPBs capture probes and SERS tags is a promising method for the detection of foodborne pathogens in food or clinical samples.

Anti-apoptotic protein BCL-XL as a therapeutic vulnerability in gastric cancer.

BACKGROUND: New therapeutic targets are needed to improve the outcomes for gastric cancer (GC) patients with advanced disease. Evasion of programmed cell death (apoptosis) is a hallmark of cancer cells and direct induction of apoptosis by targeting the pro-survival BCL2 family proteins represents a promising therapeutic strategy for cancer treatment. Therefore, understanding the molecular mechanisms underpinning cancer cell survival could provide a molecular basis for potential therapeutic interventions. METHOD: Here we explored the role of BCL2L1 and the encoded anti-apoptotic BCL-XL in GC. Using Droplet Digital PCR (ddPCR) technology to investigate the DNA amplification of BCL2L1 in GC samples and GC cell lines, the sensitivity of GC cell lines to selective BCL-XL inhibitors A1155463 and A1331852, pan-inhibitor ABT-263, and VHL-based PROTAC-BCL-XL was analyzed using (CellTiter-Glo) CTG assay in vitro. Western Blot (WB) was used to detect the protein expression of BCL2 family members in GC cell lines and the manner in which PROTAC-BCL-XL kills GC cells. Co-immunoprecipitation (Co-IP) was used to investigate the mechanism of A1331852 and ABT-263 kills GC cell lines. DDPCR, WB, and real-time PCR (RTPCR) were used to investigate the correlation between DNA, RNA, protein levels, and drug activity. RESULTS: The functional assay showed that a subset of GC cell lines relies on BCL-XL for survival. In gastric cancer cell lines, BCL-XL inhibitors A1155463 and A1331852 are more sensitive than the pan BCL2 family inhibitor ABT-263, indicating that ABT-263 is not an optimal inhibitor of BCL-XL. VHL-based PROTAC-BCL-XL DT2216 appears to be active in GC cells. DT2216 induces apoptosis of gastric cancer cells in a time- and dose-dependent manner through the proteasome pathway. Statistical analysis showed that the BCL-XL protein level predicts the response of GC cells to BCL-XL targeting therapy and BCL2L1 gene CNVs do not reliably predict BCL-XL expression. CONCLUSION: We identified BCL-XL as a promising therapeutic target in a subset of GC cases with high levels of BCL-XL protein expression. Functionally, we demonstrated that both selective BCL-XL inhibitors and VHL-based PROTAC BCL-XL can potently kill GC cells that are reliant on BCL-XL for survival. However, we found that BCL2L1 copy number variations (CNVs) cannot reliably predict BCL-XL expression, but the BCL-XL protein level serves as a useful biomarker for predicting the sensitivity of GC cells to BCL-XL-targeting compounds. Taken together, our study pinpointed BCL-XL as potential druggable target for specific subsets of GC.

Bio-barcode assay: A useful technology for ultrasensitive and logic-controlled specific detection in food safety: A review.

Food safety is one of the greatest public health challenges. Developing ultrasensitive detection methods for analytes at ultra-trace levels is, therefore, essential. In recent years, the bio-barcode assay (BCA) has emerged as an effective ultrasensitive detection strategy that is based on the indirect amplification of various DNA probes. This review systematically summarizes the progress of fluorescence, PCR, and colorimetry-based BCA methods for the detection of various contaminants, including pathogenic bacteria, toxins, pesticides, antibiotics, and other chemical substances in food in over 120 research papers. Current challenges, including long experimental times and strict storage conditions, and the prospects for the application of BCA in biomedicine and environmental analyses, have also been discussed herein.

A Rare Case of Multifocal Asynchronous Benign Granular Cell Tumors with PIK3CA Subclonal Mutation Identified in One Tumor by Next-Generation Sequencing.

Granular cell tumor (GCT) is a benign neuroectodermal tumor typically in the dermis or subcutis, although deep soft tissues and organs are occasionally involved. Multifocal GCTs are estimated to occur as many as 10% of patients. A 40-year-old female presented with multiple GCTs asynchronously involving various body sites including gastrointestinal, gynecologic, breast, urinary, and soft tissue systems. Pathologic examinations suggested benign GCTs. TruSight Tumor 170 next-generation sequencing (NGS) analysis performed on four resected tumors revealed subclonal mutation of PIK3CA p.H1047R identified in the esophageal GCT but not in the right vulva or the two cecal GCTs, suggesting that each is a primary tumor with a distinct genetic profile, rather than metastasis. PIK3CA p.H1047R is a common mutation in many cancers. Our benign GCT case demonstrates PIK3CA mutation with a low mutant allele frequency of 7%, which may represent an evolving subclone and might confer a more aggressive behavior.