The advance of the third­generation EGFR­TKI in the treatment of non­small cell lung cancer (Review).

Lung cancer is currently the second most common type of cancer with the second incidence rate and the first mortality rate worldwide. Non­small cell lung cancer (NSCLC) accounts for ~85% of the total number of cases of lung cancers. Concerning the treatment of NSCLC, targeted therapy has become a research hotspot in recent years because of its favorable efficacy, high selectivity and minimal adverse reactions. Among the drugs used in targeted therapy, the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the most common and are categorized into four generations. The use of first and second­generation drugs leads to drug resistance within 8­14 months. This resistance is primarily caused by the T790M mutation, which is the most observed mechanism. A third­generation drug has been developed to address this issue and a fourth­generation drug is expected to overcome multiple resistance mechanisms, including third­generation drug resistance. However, the fourth­generation drug has not been launched yet. At present, multiple third­generation targeted drugs have been launched globally, with three being launched in China and several being at research and clinical trial stages. The present article provides a review of the development process, mechanism of action and clinical trials of the third­generation EGFR­TKIs, aiming to provide some reference and suggestions for the clinical treatment of NSCLC and scientific research on third­generation targeted drugs.

Deciphering m6A dynamics at a single-base level during planarian anterior-posterior axis specification.

Background: The establishment of the anterior-posterior (A-P) axis is a crucial step during tissue repair and regeneration. Despite the association reported recently of N6-methyladenosine (m6A) with regeneration, the mechanism underlying the regulation of m6A in A-P axis specification during regeneration remains unknown. Herein, we deciphered the m6A landscape at a single-base resolution at multiple time points during A-P axis regeneration and constructed the de novo transcriptome assembly of the Dugesia japonica planarian. Results: Immunofluorescence staining and comparative analysis revealed that m6A is widespread across the planarian and dynamically regulated during regeneration along the A-P axis, exhibiting a strong spatiotemporal feature. The resulting datasets of m6A-modified genes identified 80 anterior-specific genes and 13 posterior-specific genes, respectively. In addition, we showed that YTHDC1 serves as the primary m6A reader to be involved in the m6A-mediated specification of A-P axis during regeneration in Dugesia japonica planarian. Conclusions: Our study provides an RNA epigenetic explanation for the specification of the A-P axis during tissue regeneration in planarian.

MAP4K4 is involved in the neuronal development of retinal photoreceptors.

Mitogen-activated protein kinase kinase kinase kinase-4 (MAP4K4) is a potential regulator of photoreceptor development. To investigate the mechanisms underlying MAP4K4 during the neuronal development of retinal photoreceptors, we generated knockout models of C57BL/6j mice in vivo and 661 W cells in vitro. Our findings revealed homozygous lethality and neural tube malformation in mice subjected to Map4k4 DNA ablation, providing evidence for the involvement of MAP4K4 in early stage embryonic neural formation. Furthermore, our study demonstrated that the ablation of Map4k4 DNA led to the vulnerability of photoreceptor neurites during induced neuronal development. By monitoring transcriptional and protein variations in mitogen-activated protein kinase (MAPK) signaling pathway-related factors, we discovered an imbalance in neurogenesis-related factors in Map4k4 -/- cells. Specifically, MAP4K4 promotes jun proto-oncogene (c-JUN) phosphorylation and recruits other factors related to nerve growth, ultimately leading to the robust formation of photoreceptor neurites. These data suggest that MAP4K4 plays a decisive role in regulating the fate of retinal photoreceptors through molecular modulation and contributes to our understanding of vision formation.

PRMT1 reverts the immune escape of necroptotic colon cancer through RIP3 methylation.

Necroptosis plays a double-edged sword role in necroptotic cancer cell death and tumor immune escape. How cancer orchestrates necroptosis with immune escape and tumor progression remains largely unclear. We found that RIP3, the central activator of necroptosis, was methylated by PRMT1 methyltransferase at the amino acid of RIP3 R486 in human and the conserved amino acid R479 in mouse. The methylation of RIP3 by PRMT1 inhibited the interaction of RIP3 with RIP1 to suppress RIP1-RIP3 necrosome complex, thereby blocking RIP3 phosphorylation and necroptosis activation. Moreover, the methylation-deficiency RIP3 mutant promoted necroptosis, immune escape and colon cancer progression due to increasing tumor infiltrated myeloid-derived immune suppressor cells (MDSC), while PRMT1 reverted the immune escape of RIP3 necroptotic colon cancer. Importantly, we generated a RIP3 R486 di-methylation specific antibody (RIP3ADMA). Clinical patient samples analysis revealed that the protein levels of PRMT1 and RIP3ADMA were positively correlated in cancer tissues and both of them predicted the longer patient survival. Our study provides insights into the molecular mechanism of PRMT1-mediated RIP3 methylation in the regulation of necroptosis and colon cancer immunity, as well as reveals PRMT1 and RIP3ADMA as the valuable prognosis markers of colon cancer.

Maintaining blood retinal barrier homeostasis to attenuate retinal ischemia-reperfusion injury by targeting the KEAP1/NRF2/ARE pathway with lycopene.

Retinal ischemia-reperfusion (I/R) often results in intractable visual impairments, where blood retinal barrier (BRB) homeostasis mediated by retinal pigment epithelium (RPE) and retinal microvascular endothelium (RME) is crucial. However, strategies targeting the BRB are limited. Thus, we investigated the inconclusive effect of lycopene (LYC) in retinal protection under I/R. LYC elevated cellular viability and reversed oxidative stress in aRPE-19 cells/hRME cells under I/R conditions based on oxygen-glucose deprivation (OGD) in vitro. Molecular analysis showed that LYC promoted NRF2 expression and enhanced the downstream factors of the KEAP1/NRF2/ARE pathway: LYC increased the activities of antioxidants, including SOD and CAT, whereas it enhanced the mRNA expression of HO-1 (ho-1) and NQO-1 (nqo-1). The activation resulted in restrained ROS and MDA. On the other hand, LYC ameliorated the damage to retinal function and morphology in a mouse I/R model, which was established by unilateral ligation of the left pterygopalatine artery/external carotid artery and reperfusion. LYC promoted the expression of NRF2 in both the neural retina and the RPE choroid in vivo. This evidence revealed the potential of LYC in retinal protection under I/R, uncovering the pharmacological effect of the KEAP1/NRF2/ARE pathway in BRB targeting. The study generates new insights into scientific practices in retinal research.

Lidocaine Inhibits Myoblast Cell Migration and Myogenic Differentiation Through Activation of the Notch Pathway.

PURPOSE: To assess the cellular and molecular effects of lidocaine on muscles/myoblasts. METHODS: Cultured myogenic precursor (C2C12) cells were treated with varying concentrations of lidocaine. RESULTS: Cell viability of C2C12 cells was inhibited by lidocaine in a concentration-dependent manner, with concentrations ≥0.08%, producing a dramatic reduction in cell viability. These ≥0.08% concentrations of lidocaine arrested cell cycles of C2C12 cells in the G0/G1 phase. Moreover, lidocaine inhibited cell migration and myogenic processes in C2C12 cells at low concentrations. Results from QRT-PCR assays revealed that following treatment with lidocaine, Notch1, Notch2, Hes1, Csl and Dll4 all showed higher levels of expression, while no changes were observed in Mmal1, Hey1, Dll1 and Jag1. CONCLUSION: This work provides the first description of the effects of lidocaine upon the regeneration of muscles and maintenance of satellite cells at the cellular and molecular levels. In specific, we found that the Dll4-Notch-Csl-Hes1 axis was up-regulated suggesting that the Notch signaling pathway was involved in producing these effects of lidocaine. These findings provide a new and important foundation for future investigations into the effects of drug therapies in muscle diseases.

2,3,5,6-Tetramethylpyrazine protects retinal photoreceptors against endoplasmic reticulum stress by modulating ATF4-mediated inhibition of PRP aggregation.

Endoplasmic reticulum (ER) stress is a common threat to photoreceptors during the pathogenesis of chronic retinopathies and often results in irreversible visual impairment. 2,3,5,6-Tetramethylpyrazine (TMP), which possesses many beneficial pharmacological activities, is a potential drug that could be used to protect photoreceptors. In the present study, we found that the cellular growth rate of 661 W cells cultured under low glucose conditions was lower than that of control cells, while the G2/M phase of the cell cycle was longer. We further found that the mitochondrial membrane potential (ΔΨm) was lower and that ER stress factor expression was increased in 661 W cells cultured under low glucose conditions. TMP reversed these trends. Visual function and cell counts in the outer nuclear layer (ONL) were low and the TUNEL-positive rate in the ONL was high in a C3H mouse model of spontaneous retinal degeneration. Similarly, visual function was decreased, and the TUNEL-positive rate in the ONL was increased in fasted C57/BL6j mice compared with control mice. On the other hand, ER stress factor expression was found to be increased in the retinas of both mouse models, as shown by reverse transcription real-time PCR (RT-qPCR) and western blotting. TMP reversed the physiological and molecular biological variations observed in both mouse models, and ATF4 expression was enhanced again. Further investigation by using western blotting illustrated that the proportion of insoluble prion protein (PRP) versus soluble PRP was reduced both in vitro and in vivo. Taken together, these results suggest that TMP increased the functions of photoreceptors by alleviating ER stress in vitro and in vivo, and the intrinsic mechanism was the ATF4-mediated inhibition of PRP aggregation. TMP may potentially be used clinically as a therapeutic agent to attenuate the functional loss of photoreceptors during the pathogenesis of chronic retinopathies. KEY MESSAGES: • Already known: TMP is a beneficial drug mainly used in clinic to enhance organ functions, and the intrinsic mechanism is still worthy of exploring. • New in the study: We discovered that TMP ameliorated retinal photoreceptors function via ER stress alleviation, which was promoted by ATF4-mediated inhibition of PRP aggregation. • Application prospect: In prospective clinical practices, TMP may potentially be used in the clinic as a therapeutic agent to attenuate the photoreceptors functional reduction in chronic retinopathies.

m6A methyltransferase METTL3 promotes retinoblastoma progression via PI3K/AKT/mTOR pathway.

Retinoblastoma (RB) is a common intraocular malignancy in children. Due to the poor prognosis of RB, it is crucial to search for efficient diagnostic and therapeutic strategies. Studies have shown that methyltransferase-like 3 (METTL3), a major RNA N (6)-adenosine methyltransferase, is closely related to the initiation and development of cancers. Nevertheless, whether METTL3 is associated with RB remains unexplored. Therefore, we investigated the function and mechanisms of METTL3 in the regulation of RB progression. We manipulated METTL3 expression in RB cells. Then, cell proliferation, apoptosis, migration and invasion were analysed. We also analysed the expression of PI3K/AKT/mTOR pathway members. Finally, we incorporated subcutaneous xenograft mouse models into our studies. The results showed that METTL3 is highly expressed in RB patients and RB cells. We found that METTL3 knockdown decreases cell proliferation, migration and invasion of RB cells, while METTL3 overexpression promotes RB progression in vitro and in vivo. Moreover, two downstream members of the PI3K/AKT/mTOR pathway, P70S6K and 4EBP1, were affected by METTL3. Our study revealed that METTL3 promotes the progression of RB through PI3K/AKT/mTOR pathways in vitro and in vivo. Targeting the METTL3/PI3K/AKT/mTOR signalling axis could be a promising therapeutic strategy for the treatment of RB.

Fractal SERS nanoprobes for multiplexed quantitative gene profiling.

Quantitative analysis is critical for biological and chemical sensing applications, yet still remains a great challenge in surface-enhanced Raman spectroscopy (SERS). Here we report the development of a novel fractal SERS nanoprobe with robust internal calibration standard and high multiplexing capability for ultrasensitive detection of DNA and microRNA. This fractal SERS nanoprobe consists of a solid Au core of ~13 nm, an inner hollow gap of ~1 nm, and a stellate outer shell. The inner hollow gap enables the embedding of Raman tags that can serve as a self-calibrating internal standard to effectively correct the fluctuations of samples and measuring conditions. The outer shell morphology is highly tunable, which provides distinct SERS enhancement and enables a reproducible quantitative measurement of nucleic acids down to femtomolar level. In addition, the flexibility of encoding crosstalk-free Raman tag molecules makes such SERS sensor particularly attractive for multiplexed bioassays. This technique is simple, reliable, and of wide applicability to various genomic screening and diagnostic applications.

Portable and Label-Free Detection of Blood Bilirubin with Graphene-Isolated-Au-Nanocrystals Paper Strip.

Point-of-care testing (POCT) devices represent a growing field that aims to develop low-cost, rapid, sensitive diagnostic testing platforms that are portable and self-contained. Surface-enhanced Raman spectroscopy (SERS) is an approach has shown high potential in POCT technology. However, the specificity or ability to uniquely detect a desired biomarker in complex biological samples is a key factor for translating SERS technologies to POCT. Herein, we fabricated cellulose SERS strips (CS) decorated with novel plasmonic nanoparticles, termed graphene-isolated-Au-nanocrystals (GIANs), for the portable detection of complex biological samples. This CS@GIANs SERS strip was used to detect free bilirubin (BR) in the blood of newborns, a biomarker of jaundice, without sample labeling or prepreparation. CS@GIANs showed superior affinity to hydrophobic BR molecules compared to typical SERS substrate, which reduced the steric hindrance effect from the nonspecific binding of BR with serum albumin in blood and improved sensitivity. Meanwhile, with the separation property of cellulose chromatography papers, CS@GIANs showed superior anti-interference to other biomolecules that had been previously adsorbed on the SERS strip. Moreover, the SERS signal from the graphitic shell of GIANs could be used as a stable internal calibration standard, which improved the reproducibility and accuracy of Raman analysis. Such a cellulose SERS strip holds high potential for enhancing current efforts in the development of rapid and low-cost point-of-care diagnostic testing.

Surfactant-Free Interface Suspended Gold Graphitic Surface-Enhanced Raman Spectroscopy Substrate for Simultaneous Multiphase Analysis.

Simultaneous multiphase detection of multiplex analytes is important, albeit challenging, especially in pharmaceuticals analysis since drugs with lipid and water solubility were often administrated together for synergistic therapy. Surface-enhanced Raman spectroscopy (SERS) is a label-free and sensitive tool for multiplex analytes detection at multiphase interfaces. However, the requirements of inducers or surfactant surface modification of the SERS substrate have restricted extensive applications. Herein, we developed a graphene-isolated-Au-nanocrystal based multiphase analysis system. Unexpectedly, the gold graphitic SERS substrate can simply suspend at the interface of the different phase without the involvement of any surfactant. Therefore, the proximity of substrate with analyte molecules remains unaffected. Such suspended substrate not only ensures sensitive SERS detection but also enables the enrichment of analytes from the different phase simultaneously without interference. Moreover, the graphitic shell of the SERS substrate has a unique vibration band located in the Raman biological silence region which is utilized as the internal standard and improves the SERS quantification accuracy. Efficient ex vivo multiphase enrichment and detection of mimic lipid- and water-soluble drugs injected into mice were demonstrated with such gold graphitic substrate, showing the potential of this simultaneous multiplex pharmacokinetic analysis.

Isotopic graphene-isolated-Au-nanocrystals with cellular Raman-silent signals for cancer cell pattern recognition.

For cancer diagnosis, technologies must be capable of molecular recognition, and they must possess a built-in pattern recognition component for efficient imaging and discrimination of targeted cancer cells. Surface enhanced Raman scattering (SERS) tags based on plasmonically active nanoparticles hold promise for accurate and efficient cancer cell recognition, owing to ultra-narrow peak and sensitive optical properties. However, a complex fingerprint spectrum increases data analysis difficulty, making it necessary to develop multicolor SERS tags with a simple fingerprint spectrum. To address this, we herein fabricated SERS-encoded nanoparticles (NPs) with stable and simple fingerprint spectrum through synthesis of isotopic cellular Raman-silent graphene-isolated-Au-nanocrystals (GIANs) and conjugation with phospholipid-polyethylene glycol-linked aptamers to target proteins overexpressed on the cancer cell surface. GIANs, which possess the properties of graphitic nanomaterials, such as super-stable optical properties and high Raman cross-section, showed enhanced SERS signals. The 2D-band Raman shift of GIAN, which located in the cellular Raman-silent region, was easily regulated through fabrication of isotopic GIANs without changing their molecular structure. Such GIAN tags demonstrated multiplexed Raman imaging capability, both in vivo and in vitro, with low background interference. Moreover, cell membrane protein (nucleolin, mucin and epithelial cell adhesion molecule)-specific, aptamer-conjugated isotopic GIANs were fabricated and feasibly applied to built-in coding for rapid imaging and pattern recognition of targeted cancer cells. Such isotopic GIAN-aptamer-encoders show high potential for efficient cancer cell identification and diagnosis.

Simultaneous Application of Photothermal Therapy and an Anti-inflammatory Prodrug using Pyrene-Aspirin-Loaded Gold Nanorod Graphitic Nanocapsules.

Photothermal therapy (PTT) has been extensively developed as an effective approach against cancer. However, PTT can trigger inflammatory responses, in turn simulating tumor regeneration and hindering subsequent therapy. A therapeutic strategy was developed to deliver enhanced PTT and simultaneously inhibit PTT-induced inflammatory response. 1-Pyrene methanol was utilize to synthesize the anti-inflammatory prodrug pyrene-aspirin (P-aspirin) with a cleavable ester bond and also facilitate loading the prodrug on gold nanorod (AuNR)-encapsulated graphitic nanocapsule (AuNR@G), a photothermal agent, through π-π interactions. Such AuNR@G-P-aspirin complexes were used for near-infrared laser-triggered photothermal ablation of solid tumor and simultaneous inhibition of PTT-induced inflammation through the release of aspirin in tumor milieu. This strategy showed excellent effects in vitro and in vivo.

Corrigendum: In situ targeted MRI detection of Helicobacter pylori with stable magnetic graphitic nanocapsules.

This corrects the article DOI: 10.1038/ncomms15653.

Graphitic nanocapsules: design, synthesis and bioanalytical applications.

Graphitic nanocapsules are emerging nanomaterials which are gaining popularity along with the development of carbon nanomaterials. Their unique physical and chemical properties, as well as good biocompatibility, make them desirable agents for biomedical and bioanalytical applications. Through rational design, integrating graphitic nanocapsules with other materials provides them with additional properties which make them versatile nanoplatforms for bioanalysis. In this feature article, we present the use and performance of graphitic nanocapsules in a variety of bioanalytical applications. Based on their chemical properties, the specific merits and limitations of magnetic, hollow, and noble metal encapsulated graphitic nanocapsules are discussed. Detection, multi-modal imaging, and therapeutic applications are included. Future directions and potential solutions for further biomedical applications are also suggested.

In situ targeted MRI detection of Helicobacter pylori with stable magnetic graphitic nanocapsules.

Helicobacter pylori infection is implicated in the aetiology of many diseases. Despite numerous studies, a painless, fast and direct method for the in situ detection of H. pylori remains a challenge, mainly due to the strong acidic/enzymatic environment of the gastric mucosa. Herein, we report the use of stable magnetic graphitic nanocapsules (MGNs), for in situ targeted magnetic resonance imaging (MRI) detection of H. pylori. Several layers of graphene as the shell effectively protect the magnetic core from corrosion while retaining the superior contrast effect for MRI in the gastric environment. Boronic-polyethylene glycol molecules were synthesized and modified on the MGN surface for targeted MRI detection. In a mouse model of H. pylori-induced infection, H. pylori was specifically detected through both T2-weighted MR imaging and Raman gastric mucosa imaging using functionalized MGNs. These results indicated that enhancement of MRI using MGNs may be a promising diagnostic and bioimaging platform for very harsh conditions.

Stable Graphene-Isolated-Au-Nanocrystal for Accurate and Rapid Surface Enhancement Raman Scattering Analysis.

Various interferences from measurement conditions and substrate inhomogeneity are well-known confounding factors for poor reproducibility, which is a challenge in surface-enhanced Raman scattering (SERS) quantification. To address these issues, novel substrates and versatile internal standards have been designed and the repeatability is improved to some degree. However, these internal standards are either complex or unstable enough to resist harsh environments such as acid and oxidation. Graphene-isolated-Au-nanocrystal (GIAN) has unique properties and been applied for cell multimodal imaging and chemotherapy but not for SERS quantification analysis yet. Herein, we chose GIANs to improve the accuracy of SERS analysis. GIAN integrates the SERS effect and internal standard into a simple nanoparticle and is proved to be an ideal platform for SERS analysis given its superior properties: (1) chemical stability, it remains stable in strong acid and oxidation, even mimic bioenvironment; (2) a simple core-shell structure, with a thin graphitic shell which is not only a protector that avoiding inner Au catalysis unnecessary reaction but also an internal standard to eliminate the interference during the Raman detections; (3) the big-Π structure can absorb target molecule thus achieve an enrichment effect and quench background fluorescence. Laser power, focus, and substrate fluctuations as well as coexist substance interferences were investigated and the accuracy was improved greatly with the introduction of 2D band internal standard in Raman silent region with less background. Moreover, GIAN was applied for crystal violet determination directly on fish muscle and scale, which was rapid and convenient without complex extraction process. All these results indicate GIAN is an optimum choice for SERS analysis in complex systems.

Elucidating the cellular uptake mechanism of aptamer-functionalized graphene-isolated-Au-nanocrystals with dual-modal imaging.

Elucidating the endocytosis and metabolism of nanoparticles in cells could improve the diagnostic sensitivity and therapeutic efficiency. In this work, we explore the cellular uptake mechanism of a biocompatible nanocrystal nanostructure, graphene-isolated-Au-nanocrystals (GIANs), by monitoring the intrinsic Raman and two-photon luminescence signals of GIANs in live cells. Aptamers functionalized on the GIAN nanostructure through simple, but strong, π-π interactions entered the cells through a clathrin-dependent pathway, while unmodified GIANs mainly entered the cells through a caveolae-mediated endocytosis pathway. Thus, it can be concluded that the mechanism of cellular uptake in these graphene-isolated-Au-nanocrystal nanostructures is determined by the presence or absence of aptamer modification.

Modulating the Morphology of Gold Graphitic Nanocapsules for Plasmon Resonance-Enhanced Multimodal Imaging.

With their unique optical properties and distinct Raman signatures, graphitic nanomaterials can serve as substrates for surface-enhanced Raman spectroscopy (SERS) or provide signal amplification for bioanalysis and detection. However, a relatively weak Raman signal has limited further biomedical applications. This has been addressed by encapsulating gold nanorods (AuNRs) in a thin graphitic shell to form gold graphitic nanocapsules. This step improves plasmon resonance, which enhances Raman intensity, and has the potential for integrating two-photon luminescence (TPL) imaging capability. However, changing the morphology of gold graphitic nanocapsules such that high quality and stability are achieved remains a challenge. To address this task, we herein report a confinement chemical vapor deposition (CVD) method to prepare the construction of AuNR-encapsulated graphitic nanocapsules with these properties. Specifically, through morphological modulation, we (1) achieved higher plasmon resonance with near-IR incident light, thus achieving greater Raman intensity, and (2) successfully integrated two-photon luminescence dual-modal (Raman/TPL) bioimaging capabilities. Cancer-cell-specific aptamers were further modified on the AuNR@G graphitic surface through simple, but strong, π-π interactions to achieve imaging selectivity through differential cancer cell recognition.

Simultaneous tracking of drug molecules and carriers using aptamer-functionalized fluorescent superstable gold nanorod-carbon nanocapsules during thermo-chemotherapy.

Controlling and monitoring the drug delivery process is critical to its intended therapeutic function. Many nanocarrier systems for drug delivery have been successfully developed. However, biocompatibility, stability, and simultaneously tracing drugs and nanocarriers present significant limitations. Herein, we have fabricated a multifunctional nanocomposite by coating the gold nanorod (AuNR) with a biocompatible, superstable and fluorescent carbon layer, obtaining the AuNR@carbon core-shell nanocapsule. In this system, the carbon shell, originally obtained in aqueous glucose solutions and, therefore, biocompatible in physiological environments, could be simply loaded with cell-specific aptamers and therapeutic molecules through π-π interactions, a useful tool for cancer-targeted cellular imaging and therapy. Moreover, such a stable and intrinsic fluorescence effect of the AuNR@carbon enabled simultaneous tracking of released therapeutic molecules and nanocarriers under thermo-chemotherapy. The AuNR@carbons had high surface areas and stable shells, as well as unique optical and photothermal properties, making them promising nanostructures for biomedical applications.