Therefore, there is an urgent call to translate these preclinical results for nanoprobes into clinical trials. For this reason, this review aims to give an overview of currently investigated nanoprobes in the context of atherosclerosis, summarize relevant published studies showing applications of different kinds of formulated nanoprobes in early detection and reverse of plaques, discuss recent advances and some limitations thereof, and provide some insights into the development of the new generation of more precise and efficient molecular nanoprobes, with a critical property of specifically targeting early atherosclerosis.Atherosclerosis, an inflammation-driven chronic blood vessel disease, is a major contributor to devastating cardiovascular events, bringing serious social and economic burdens. Currently, non-invasive diagnostic and therapeutic techniques in combination with novel nanosized materials as well as established molecular targets are under active investigation to develop integrated molecular imaging approaches, precisely visualizing and/or even effectively reversing early-stage plaques. Besides, mechanistic investigation in the past decades provides many potent candidates extensively involved in the initiation and progression of atherosclerosis. Recent hotly-studied imaging nanoprobes for detecting early plaques mainly including optical nanoprobes, photoacoustic nanoprobes, magnetic resonance nanoprobes, positron emission tomography nanoprobes, and other dual- and multi-modality imaging nanoprobes, have been proven to be surface functionalized with important molecular targets, which occupy tailored physical and biological properties for atherogenesis. Of note, these engineering nanoprobes provide long blood-pool residence and specific molecular targeting, which allows efficient recognition of early-stage atherosclerotic plaques and thereby function as a novel type of precise diagnostic tools as well as potential therapeutic carriers of anti-atherosclerosis drugs. There have been no available nanoprobes applied in the clinics so far, although many newly emerged nanoprobes, as exemplified by aggregation-induced emission nanoprobes and TiO Atherosclerosis, a predominant cause of death and disability worldwide, is well characterized by excessive smouldering inflammation, and lipid metabolism dysfunction in terms of the pathological nature In fact, there are quite many clinical techniques applied for atherosclerotic plaque monitoring, as exemplified by computed tomography angiography (CTA), contrast-enhanced cardiac computed tomography (CT), intravascular ultrasound (IVUS), etc. in vivo, eventually leading to early diagnosis of atherosclerosis and other cardiovascular diseases, improved treatment strategies, and accurate assessment of treating efficacy.Among various molecular probes, nanoprobes, as a novel type of imaging probes and ultrasmall biosensors, have been shown to contribute to the early diagnosis of multiple diseases, such as Alzheimer's disease Based on this background information, this review aims to give a comprehensive overview of currently-studied nanoprobes in the context of atherosclerosis, summarize published experimental studies showing detective and therapeutic effects of different kinds of engineering nanoprobes on early plaques, go through the contribution of some molecular targets to adding specificity of nanoprobes and discuss current progress and some limitations thereof. Through looking into four common types of single-modality nanoprobes, some dual- and multi-model nanoprobes, and potent molecular targets for early-stage plaque detection and reverse, such as inflammatory mediators and lipid metabolism-related factors, some insights have been gained into the design of more precise and efficient nanoprobes for the recognization of plaque-specific molecules, to achieve early diagnosis and prevention of atherosclerosis eventually.With regard to the application of nanoprobes in targeting plaques, we would like to start with the contribution of molecular imaging in this context. As a non-invasive strategy, molecular imaging mainly targets specific molecules on the tissue and cell levels, and especially shows their changes in the pathological states, in order to have an in-depth understanding of the disease mechanisms, develop new pharmacological targets, and provide novel drug candidates Proposed applications of engineering nanoprobes in atherosclerosis are mainly divided into two types. First of all, various preclinical studies have shown that formulated nanoprobes can specifically recognize early-stage plaques through targeting plaque-specific molecules. Detection of nanoprobes can be accomplished by a variety of methods. Moreover, the successful imaging of monocytes, macrophages, foam cells, and other plaque components, has enabled these nanoprobes to become promising in realizing the visualization of plaques, especially in the early stage of atherosclerosis More interestingly, new dual-mode imaging formed by the fusion of multiple ones, such as optical/MR dual-model imaging, optical/ultrasound dual-modality imaging, and photoacoustic/ultrasound imaging and others, has attracted wide attention and tested for atherosclerotic animal models Table 1, covering their major advantages as well as disadvantages. Currently investigated single-modality molecular nanoprobes for plaque detection mainly include optical imaging nanoprobes, photoacoustic imaging nanoprobes, magnetic resonance imaging nanoprobes, and positron emission tomography imaging nanoprobes, which have been summarized in Table 2 and Figure 1, and will be discussed in the following subchapters. Moreover, newly emerging dual-modality imaging nanoprobes will be described in the next section, and summed up in Table 3 and Figure 2. Taken together, these theranostic applications of these single- and/or dual-modality imaging nanoprobes in atherosclerosis, demonstrated by many recent original studies, would give more hope for early diagnosis and prevention of patients with plaques in clinical practice.To the best of our knowledge, there has been no systematic review reported for recent advances in formulating nanoprobes in the context of atherosclerosis so far. In this review, comparisons of different kinds of nanoprobes for atherosclerotic plaques have been listed in In the scope of optical imaging, fluorescent materials for labeling, including nano fluorescent probes, have been widely applied to develop optical imaging technology, providing new approaches for early monitoring and treatment of some diseases, such as cancers Apolipoprotein E-deficient (-/-Apoe) mice et al. have applied CD36-antibody-modified-luminescence nanoprobes for in situ imaging of CD36 activation as well as CD36-ox-LDL binding in plaque-associated macrophages with high sensitivity and good stability, which has not been directly visualized in living macrophages by other imaging tools Wang and colleagues have recently developed a kind of highly bright aggregation-induced emission (AIE) nanoprobes, which are designed to functionalize with anti-cluster of differentiation (CD) 47 antibodies, to detect early-stage plaques in et al. have tested an MMP-2-specific aptamer-conjugated fluorescent nanoprobe in -/-Apoe mice to visualize atherosclerotic plaques ex vivo imaging. It is worth mentioning that this nanoprobe may be available not only for atherosclerotic plaque imaging, but also for gastric cancer tissue visualization. Because MMP-2 aptamer could detect MMP-2 expression in both types of tissues in vivo imaging of senescent vascular cells in atherosclerotic mice in vivo imaging of senescent cells in pathological vasculatures, although there are several fluorescent probes reported due to the overexpression of senescence-associated β-galactosidases in senescent cells -/-Apoe mice Given the fact that matrix metalloproteinase-2 (MMP-2) contributes to atherogenesis through affecting the functional activities of immune cells, endothelial cells, vascular smooth muscle cells (VSMCs), and platelets in vivo and in vitro. Even though these formulated nanoprobes are still limited to preclinical exploration, current preliminary data provide a solid basis for their clinical application. In addition to these molecular targets discussed in the above paragraphs, other key players in atherogenesis, such as inflammatory mediators, classical and atypical chemokines, receptors, and lipid metabolism-related factors should be also included in future studies, which would offer more choices for designing more sensitive and feasible nanoprobes under the guidance of advanced imaging systems.In summary, optical imaging nanoprobes are one type of the most commonly-studied nanoprobes for atherosclerotic plaque detection. So far, CD47, CD36, β‐galactosidase, MMP-2, and osteopontin have been targeted and further validated both Table 2.As a non-invasive and non-ionizing biomedical imaging method, photoacoustic imaging (PAI) technology, combining the acoustic resolution and imaging depth of ultrasonography with the sensitivity of optical imaging, has been well developed and applied in the field of atherosclerosis research in the past decade 2S PAI nanoprobes for plaque imaging in -/-Apoe mice, showing relatively long blood retention as well as selective accumulation in plaques due to the lipophilicity of the C18 chain to the atherosclerotic microenvironment 2S PAI nanoprobes have good hemocompatibility and no side effects on the main organs, as shown by hemolysis and coagulation assays. These results together support this fabricated nanoprobe as an available noninvasive imaging tool for atherosclerotic plaques in vivoet al. and Ge et al. have developed two kinds of nanoprobes to visualize vulnerable atherosclerotic plaques (VASPs) et al. used the self-assembly of bovine serum albumin (BSA), to construct a kind of BSA-Cy-Mito nanoprobe as a GSH/H2O2 indicator for in vivo photoacoustic imaging of redox status in ox-LDL-activated macrophages as well as high fat diet-fed -/-Apoemice, in order to evaluate VASP formation with high accuracy. According to their redox states among different types of plaques, BSA-Cy-Mito nanoprobes could specifically distinguish vulnerable and stable plaques, indicating this sensitive redox-responsive PAI nanoprobe may act as a powerful tool for early identification of rupture-prone plaques et al. also focused on features of VASPs using another type of nanoprobes, i.e. formulated osteopontin (OPN) Ab/Ti3C2/ICG nanoprobes. In addition to the differentiation of VASPs, OPN Ab/Ti3C2/ICG nanoprobes also highlight the importance of OPN in the non-invasively specific imaging of VASPs at the molecular level Wu and coworkers have reported a type of ICG@PEG-Aget al. and described in the above subchapter, Xie et al. also target CD36 and identify the CD36 positive expression as the inflammation level 2-HA-p nanoprobes have been reported as a classical example of metal-based nanoprobes applied in experimental studies for atherosclerosis on the basis of PAI 2-HA-p nanoprobe-elicited mild phototherapy leads to decreased intracellular lipid levels in foam cells, mechanistically through the regulation of the SREBP2/LDLR pathway as well as ABCA1-mediated cholesterol efflux. This study clearly addresses the therapeutic potential of black TiO2-HA-p nanoprobes in atherosclerosis Besides, Xie and colleagues have applied a kind of semiconducting polymer nanoparticle-based probe, to specifically identify inflammatory components involved in atherogenesis and further evaluate the inflammation severity by utilizing atherogenic mouse models. Consistent with experimental findings obtained via luminescence nanoprobes formulated by Sun As demonstrated by the above several studies, PAI nanoprobes have been popularly investigated in the context of atherogenesis. With the advantages of optical imaging and ultrasonography, nanoprobe-based PA imaging is a preferable solution to screen the critical ingredients of atherosclerotic plaques at the molecule level, providing many opportunities for further exploring novel noninvasive imaging techniques of deeper tissues, such as human deeper coronary arteries. Targeting MMP-2, osteopontin, CD36, etc., would further improve the specificity of these PAI nanoprobes, which helps to differentiate advanced vulnerable plaques from early-stage lesions on the basis of the image pattern and the degree of contrast enhancement. However, we have to acknowledge that optical/photoacoustic imaging nanoprobes have been only applied in cell lines and experimental animals due to some limitations of the living biological imaging system. They are often enriched in the liver and difficult to metabolize, which leads to strong background signals and poor imaging quality, preventing them from entering the clinic.To date, atherosclerotic plaques and their major components, as exemplified by pro-inflammatory macrophages, have also been extensively characterized by magnetic resonance imaging (MRI), which can generate images with high spatial resolution and excellent soft-tissue contrast et al. designed EGFP-EGF1-SPIONs as TF-targeted magnetic nanoprobes to precisely and specifically detect TF-expressing cells, such as monocytes/macrophages, endothelial cells, and/or smooth muscle cells in atherosclerotic plaques, which may support to monitor the incidence of early cardiovascular and cerebrovascular events driven by rupturing plaques et al. have formulated Fe3O4@M nanoprobes by coating Fe3O4 biomimetic nanoparticles with the macrophage membrane, which can effectively target early atherosclerotic lesions by the specific recognition of integrin α4β1 to vascular cell adhesion molecule-1 (VCAM-1) 3O4@M nanoprobes, on the other hand, supports specifically recognizing and binding to VCAM-1 through their highly expressed α4β1 integrin. These features together ensure Fe3O4@M nanoprobes as a suitable imaging tool for early plaque detection Given the fact that TF is a key proatherogenic factor et al. have developed a kind of gadolinium immunonanoparticle-based nanoprobe via targeting inflammation-associated myeloid-related protein (MRP) 8/14, which is an extracellularly secreted protein involved in atherogenesis -/-Apoemice in vivo111In3+ radiolabeled probes were found to bind to the macrophage scavenger receptor A (SR-A) with high-affinity through in vitro characterizations. Additionally, higher levels of surface sulfation lead to much higher uptake efficiency by macrophages, as shown by cell uptake studies, revealing a new standard metric for targeted nanomaterials Moreover, Maiseyeu To sum up, MRI nanoprobes have shown good prospects for the early diagnosis and treatment of experimental atherosclerosis, even though there are several disadvantages, for example, small molecular weight, short half-life, and high toxicity of some molecular probes. Even so, in comparison to optical/photoacoustic nanoprobes, MRI nanoprobes have a higher possibility to achieve clinical translation, without limitations of equipment. We believe recently-investigated MRI nanoprobes conjugated with different molecular targets would provide more choices for specific recognization and comprehensive evaluation of early-stage plaques.18F-FDG) lacks specificity for certain cell types, which can be improved by using nanoparticle-based PET imaging agents that has good targeting properties for atherosclerotic plaques.Positron emission tomography (PET) is particularly suitable for the non-invasive and quantitative characterization of macrophage-mediated inflammation, vascular calcifications as well as angiogenesis in atherosclerosis owing to the high tissue penetration and superior sensitivity ex vivo and in vivo, and further addressed its potential for clinical translation So far, there are several studies to apply PET imaging nanoprobes in atherosclerotic research, including single-modality as well as dual-modality nanoprobes. In this section, single-model PET imaging nanoprobes are mainly introduced here. Given that angiogenesis is a complex biologic process in atherosclerosis et al. developed 89Zr-HDL nanoparticles in combination with noninvasive PET imaging, to visualize its accumulation in advanced plaques. Of special interest, this formulating nanoprobe has been validated in different kinds of atherosclerotic animal models, such as -/-Apoe mice, rabbits, and pigs 64Cu-CANF-comb nanoprobes to assess the in vivo PET imaging of NPR-C, which is expressed on atherosclerotic plaques, and found that the 25% 64Cu-CANF-comb shows the best NPR-C targeting specificity as well as sensitivity in -/-Apoemice, suggesting the 25% 64Cu-CANF-comb as a good PET imaging agent to detect atherosclerosis Considering that high-density lipoprotein (HDL) is a natural nanoparticle that interacts with macrophages in atherosclerotic plaques, Pérez-Medina Table 3 and Figure 2 , have also been discussed in this section.To date, dual-modality imaging nanoprobes have been becoming popular, as single-mode imaging is not enough to collect accurate imaging information of plaques. Of note, dual- or multi-modality simultaneous imaging techniques facilitate the integration of information on both anatomy and function, and thus have the potential to improve diagnostic and prognostic evaluation for atherosclerosis 3O4 nanoparticle-based probes, and introduced in this subchapter as well et al. have applied osteopontin-targeted probes based on Fe3O4 nanoparticles to achieve MRI/optical dual-modality imaging of VASPs 3O4 nanoparticle-based probes have low toxicity, which makes them available in the noninvasive evaluation of early plaques. In their study, these formulated nanoprobes have been found to specifically recognize foamy macrophages through in vitro cell experiments, and further visualize vulnerable plaques as demonstrated by -/-in vivo Apoe mouse model studies In addition to the application of single optical imaging, the hybrid optical/MR imaging system is also involved in atherosclerotic research. MRI is applied to collect the anatomical information of the diseased blood vessels, and optical fluorescence imaging can further obtain cellular and molecular information due to its high sensitivity -/-Apoe mice Owing to that macrophages are key components of atherosclerotic plaques, several researchers have developed dual-modality nanoprobes using molecular targets related to macrophage activation, which would be discussed in detail in this section. Yao and colleagues targeted folate receptor (FR)-β, and formulated a dual-modal fluorescent/MRI contrast agent to detect inflammation-associated activated macrophages within carotid plaques et al. developed profilin-targeted magnetic nanoparticles, i.e. PC-NPs, as dual-modality molecular probes for murine atherosclerosis, and revealed their potential in characterizing VSMCs in plaques. Importantly, a good correlation between MRI signals and fluorescence intensities of imaging in-/- Apoe mice with PC-NPs injection was observed et al. submitted that the ROS-scavenging nanoparticles can mediate MR/fluorescence dual-modality imaging tracing of vulnerable plaques. It is worth pointing out that this is the only study to especially address the therapeutic potential of dual-modality nanoprobes in atherosclerosis, mechanistically through the downergulation of inflammation, apoptosis, and foam cell formation In addition to macrophage activation, Fang and coworkers mainly looked into biomarkers associated with the angiogenesis process, and eventually achieved dual-modality imaging of unstable plaques via utilizing VEGFR2-targeted upconversion nanoprobes. Of note, FITC-VRBP1 has been observed to bind to HUVECs with high specificity. Furthermore, the successful optical/MR dual-modality imaging targeting angiogenesis in plaques has been obtained through applying VRBP1-UCNPs, confirming that it is a promising technique to detect unstable plaques in early-stage Among various diagnostic imaging techniques, ultrasound imaging has its own advantages for atherosclerotic plaques, for example, real-time monitoring capability, low cost, portability, and high safety et al. constructed RGDfk peptide-targeted nanoprobes to study angiogenesis in atherosclerotic plaques, not only obtaining functional information of multiple components in plaques, such as neovascularization and lipid core, but also acquiring arterial structural information. In their study, an atherosclerotic rabbit model was utilized, and the imaging effects of photoacoustic/ultrasound dual-modality imaging nanoprobes on plaques were systematically evaluated in vivoex vivo coronary artery plaque photoacoustic/ultrasound imaging in a patient undergoing autopsy by using silica-coated gold nanorods as contrast agents For many years, an integrated intravascular imaging catheter has been successfully designed and developed, making it possible for the clinical realization of the hybrid intravascular ultrasound/intravascular photoacoustic (IVUS/IVPA) imaging system et al. applied 64Cu-DOTA-DAPTA-comb and 64Cu-vMIP-II-comb nanoprobes in different studies, respectively, and found that plaque progression was in line with increased expression of chemokine receptors as well as elevated PET signals. These results together indicate the potential of PET/CT imaging nanoprobes in detecting plaque progression in C57BL/6 mice and/or -/-Apoe mice 64Cu-DAPTA-comb nanoprobes could visualize lesions by detecting CCR5 expression with high sensitivity and specificity Due to its high sensitivity and quantitative diagnosis, the integrated approach of PET/CT has been widely studied in preclinical and clinical atherosclerotic research among various integrated imaging modalities et al. recently et al. utilized [68Ga]Pentixafor PET/MR imaging to evaluate the expression of CXCR4 in human atherosclerotic lesions, highlighting CXCR4 as a surrogate marker for atherosclerosis Nowadays, PET/MR dual-modality imaging has been extensively applied to study inflammation in atherosclerosis by mapping key molecular processes as well as functional parameters, as reviewed by Senders 64Cu-labeled dextran sulfate-coated iron oxide nanoparticles, and thereby mapped macrophage distribution in lesion area using PET/MR imaging. Then, the enhanced contrast induced by these nanoprobes was observed at sites of vascular inflammation, but not in a normal vessel, in both PET and MR images et al. developed a kind of multifunctional PET/MRI nanoprobe, i.e. 64Cu-NOTA-IONP@MMP2c-PEG2K, MMP2cNPs, to evaluate the expression of MMP-2 in macrophage-rich vascular lesions. Interestingly, the excellent plaque-to-normal carotid artery contrast was obtained due to the rapid clearance of MMP2cNPs from the contralateral normal carotid artery. Moreover, iron was accumulated in atherosclerotic plaques, which was colocalized with MMP-2 in macrophages, as confirmed by histological analyses Jarrett and colleagues synthesized et al. mainly looked into angiogenesis in a rabbit atherosclerotic model via using GEBP11 peptide-targeted magnetic iron oxide nanoparticles, showing good imaging properties, high stability, and little cytotoxicity as well as low immunogenicity, in the context of PET/MR dual-modality imaging 68Ga iron oxide nano-radiomaterials for the targeted bioorthogonal molecular imaging, with their selective accumulation in atherosclerotic plaques in -/-Apoe mice et al. reported hydroxyapatite (HAP)-multitag as a PET contrast nano-tracer for the characterization of vascular calcifications in plaques, and revealed that HAP-multitag can support to achieve the early detection of plaques in 16-week-old -/-Apoe mice. Importantly, these imaging probes are capable of providing simultaneous signals in both modalities, i.e. PET/MRI In contrast, Su 64Cu-TNP, and observed a pronounced correlation between PET signals and CD68 expression. Of interest, they addressed the diagnostic capability of multi-modality nanoprobes in atherosclerosis and their clinical translatability as well et al. developed a novel multimodal imaging agent, 5-HT-Fe3O4-Cy7 nanoparticles, for MRI, CT, and fluorescence imaging of vulnerable plaques by detecting active myeloperoxidase, displaying high sensitivity and specificity. The severity of inflammation and the activity of myeloperoxidase could be evaluated according to the accumulation of these nanoprobes in plaques Not only high-sensitivity imaging techniques but also specific targeting markers are needed for an ideal imaging platform to identify atherosclerotic plaques precisely and specifically. Therefore, developing multi-modality imaging nanoprobes is an appealing trend in the future, although there might be some technical challenges in constructing nanomaterials with tailored physical and biological properties. In fact, various multi-modality imaging has been applied to study tumorigenesis Table 4 as well as Figure 2.Considering the importance of molecular targets in determining the specificity of nanoprobes for plaque visualization, we would discuss currently-applied targets and some other potent candidates in this section, separately. In the meanwhile, relevant details and comparison of several well-investigated molecular targets, encompassing VCAM-1, OPN, MMP-2, CD47, CD44, MARCO, and VEGFR-2, etc., have been summarized in Current plaque-relevant molecular targets that have been chosen for the design of nanoprobes, mainly include aptamers, peptides, antibodies, and others. In general, antibodies have high specificity and affinity, whereas high immunogenicity and poor stability. Additionally, high cost is also a disadvantage limiting antibodies' application. In contrast, peptides need relatively low production costs, with high specificity and safety but limited stability Table 4, these targets are closely associated with different biological processes in atherogenesis, for example, inflammation, angiogenesis, vascular calcifications, and lipoprotein synthesis as well as clearance On the other hand, as shown in in vitro and experimental animals in vivo, due to some limitations of the living biological imaging system, as exemplified by fluorescent nanoprobes. Especially, they are often enriched in the liver and difficult to metabolize, which leads to strong background signals and poor imaging quality. Although these shortcomings prevent their clinical application, they can still be utilized to study the pathological mechanisms of atherosclerosis, and evaluate the drug efficacy. In contrast to optical/photoacoustic nanoprobes, MR/PET nanoprobes have a higher likelihood to achieve preferable clinical translation once their specificity and selectivity are improved, without limitations of equipment.In recent years, molecular imaging techniques that enable early monitoring of atherosclerotic plaques before clinical manifestation is an active area of research. Various single-, dual- as well as multi-modality imaging nanoprobes have been investigated and show promising prospects. However, optical/photoacoustic imaging nanoprobes have been only applied in cell lines In this regard, it is worth noting that there are several potential factors preventing these nanoprobes from entering the clinic, as implied by many original studies. First of all, targeting efficiency is a key issue. Owing to the limited specificity of current nanoprobes in detecting plaques, target-specific ligands are extremely needed to further improve the accuracy of the molecular diagnosis of early atherosclerosis. Nowadays, researchers have developed various types of nanoprobes coated with well-studied plaque-specific molecules, exerting tailored physical and biological properties. They on the one hand are beneficial to specific recognization of early-stage plaques, on the other hand, give additional insights into the molecular basis for atherogenesis. However, the presence of the same receptor in different cells still reduces the targeting capacity of the molecular probes, in turn limiting its clinical application and drawing more attention from researchers. Secondly, we have to consider the metabolic issues of nanomaterials when it comes to their application in patients. Recently, metabolizable near-infrared-II nanoprobes seem to be a better choice to overcome this drawback. Thirdly, the complexity and the required dosage of these nanoprobes for imaging need to be optimized to achieve the medical purpose, which is also one obvious limitation. Thus, these key factors should be taken into account when designing novel nanoprobes in the context of atherosclerosis.In addition, dual- and multi-mode imaging combining the advantages of multiple single-mode imaging techniques become more popular, as single-modality imaging is not enough to provide accurate imaging information. For this reason, developing dual- and multi-modality nanoprobes and/or combining multiple molecular targets in one type of nanoprobe is the common trend in this field. Furthermore, rigorous validation of the signal origin and the probe fate would be guaranteed by the multi-modality capabilities of these nanomaterials. Undoubtedly, specific targets, metabolizable nanomaterials, and advanced equipment, are together to support the precise visualization of plaques. Of interest, cell membrane-coated nanotechnology has emerged as a promising therapeutic platform, which may facilitate the targeted delivery of anti-atherosclerosis drugs. In the future, multi-modality nanoprobes with multiple targets would strongly contribute to accurate diagnosis and efficient treatment of atherosclerosis, although their complexity may bring some technical difficulties.In summary, this paper reviews different types of formulated nanoprobes applied for early detection and reverse of plaques, and especially highlights recent advances, many challenges as well as opportunities. Now clearly, with the development of metabolizable nanomaterials and the presence of more specific targets, the clinical value of nanoprobes with the critical property of specifically targeting early atherosclerosis has been increasing. Of special note, the new generation of more precise and efficient molecular nanoprobes also needs the close collaboration of cardiologists, chemists, clinical radiologists, radiobiologists, statisticians, molecular biologists, and immunologists. Taken all together, engineering nanoprobes, serving as precise diagnostic tools and promising therapeutic carriers, would provide more preferable choices for early diagnosis and prevention of atherosclerosis.


The transplant community has faced unprecedented challenges balancing risks of performing living donor transplants during the COVID-19 pandemic with harms of temporarily suspending these procedures. Decisions regarding postponement of living donation stem from its designation as an elective procedure, this despite that the Centers for Medicare and Medicaid Services categorise transplant procedures as tier 3b . In times of severe resource constraints, health systems may be operating under crisis or contingency standards of care. In this manuscript, the United Network for Organ Sharing Ethics Workgroup explores prioritisation of living donation where health systems operate under contingency standards of care and provide a framework with recommendations to the transplant community on how to approach living donation in these circumstances.To guide the transplant community in future decisions, this analysis suggests that: (1) living donor transplants represent an important option for individuals with end-stage liver and kidney disease and should not be suspended uniformly under contingency standards, (2) exposure risk to SARS-CoV-2 should be balanced with other risks, such as exposure risks at dialysis centres. Because many of these risks are not quantifiable, donors and recipients should be included in discussions on what constitutes acceptable risk, (3) transplant hospitals should strive to maintain a critical transplant workforce and avoid diverting expertise, which could negatively impact patient preparedness for transplant, (4) transplant hospitals should consider implementing protocols to ensure early detection of SARS-CoV-2 infections and discuss these measures with donors and recipients in a process of shared decision-making. Living donor kidney and liver transplantation represents the optimal treatment for individuals with end-stage kidney and end-stage liver disease. These life-saving therapies are often contextualised as elective procedures because the donor operation should be conducted in way as to not pressure participation in any manner, yet the transplant procedure in the recipient may have medical urgency based on patient need. The stress on hospital systems due to the COVID-19 2020 Spring surge was unprecedented and legitimate concerns regarding exposure risk, inadequate testing protocols, personal protective equipment and intensive care unit availability required rationing of resources and prioritisation in the types of care delivered.functionally equivalent to usual patient care.Immense demands of patients with COVID-19 continue to tax health systems, forcing them to transition to ‘contingency standards of care’ and plan for ‘crisis standards of care’. Crisis standards of care is defined as ‘a substantial change in usual healthcare operations and the level of care it is possible to deliver, which is made necessary by a pervasive  or catastrophic  disaster’.This ethical analysis examines whether proceeding with the living donor transplantation is appropriate when the goal is to provide contingency standards of care—not crisis standards—as long as there are safeguards in place that minimise exposure risk, maintain staff expertise and incorporate engagement of donor/recipient pairs in decisions about their willingness to accept often unquantifiable exposure risks. The aim is not to question prior decisions made under extraordinary circumstances, rather to learn from previous experiences and develop comprehensive decision-making tools to guide actions during future circumstances where healthcare resources are stressed.instrumental value may have different applications in bioethics, but as used in times of rationing healthcare refers to prioritising those—such as living donors—who have the means to save others.manner in which healthcare ends are procured is typically better understood instrumentally .In determining what priority to assign living donor transplantation, particularly in times of rationing, both intrinsic and instrumental value considerations become relevant. An intrinsic value refers to a normative preference that is inherently appreciated for its own sake, while an instrumental value is prudential, or strategic, and, thus, seeks to achieve a further end. The term Living donor transplantation has obvious intrinsic value, both for the recipient waiting for transplant, and for the living donor who has resolved to donate his, her, or their organ for a recipient’s benefit. At the same time, there are instrumental values to consider in performing living donor transplantation in times of severe resource constraints, where the clear benefits to society of proceeding with a planned transplant must be balanced against critical resources being exigently distributed in other ways. In this case, there are a number of instrumental values to consider. For example, does our allocation of resources appropriately balance the interests of patients of specific disease state, like end-stage organ failure, with the needs of the population-at-large? For example, there were other clinical conditions, for example, cancer patients, who were also subjected to delays in treatment, supply chain disruptions that impacted care and postponement of operative procedures, particularly in situations where radiotherapy or chemotherapy were considered reasonable alternatives.In considering the prioritisation of living donation in such circumstances, the pragmatism of preserving existing relationships between healthcare providers must also be instrumentally evaluated, for example, between dialysis centres and transplantation centres. There are different levels of scarcity, capacity and expertise that need to be considered and which are likely to be fluid requiring constant re-evaluation. For example, a living donor recipient who is predicted to require an ICU bed may or may not be an appropriate candidate dependent on several health system factors that could change in a short amount of time.Ideally, we would pursue a policy that reconciles intrinsic values with our understanding of the greater good, and where instrumental considerations align and reinforce intrinsic goals. In considering how intrinsic values cohere with instrumental values, during times of crisis, it may be useful to identify critical thresholds beyond which we revisit standing policies in order to make sure that all of the relevant considerations are being taken into account. These types of value assessments require a determination of a given need and the social context of a donation. These can only be fully understood through a framework that supports an open dialogue with patients, their respective donors and considerations of the potential impact to non-transplant patients whose care may be affected.The rationing of healthcare in times of resource scarcity is both required and ethical.The ethical principles that govern organ transplantation should ideally balance considerations of utility, justice (equity) and respect for persons (autonomy).In Spring 2020, 81% of transplant centres in regions of the country with a high cumulative COVID-19 prevalence (≥500 cases/100k population) chose to internally suspend their living donor kidney programmes, although none formally inactivated their programmes with Organ Procurement and Transplantation Network/United Network for Organ Sharing.et al have analysed data from the Scientific Registry of Transplant Recipients on the early effect of COVID-19 on kidney transplant outcomes. They show that new listings, deceased donor transplants and living donor transplants declined by 18%, 24% and 87%, respectively (15 March 2020 to 30 April 2020). States with the highest rates of COVID-19 cases had the lowest rates of living kidney donor transplants than expected (IRR=0.000.010.05), while these same states had a 2.2-fold higher observed waitlist mortality (IRR=1.882.222.62).Boyarsky The first surge was associated with a decrease in total living donor kidney transplants performed compared with 2019, while deceased donor volume was largely unaffected . This coet al also make the point that cessation of living donor transplant is particularly relevant in areas where dialysis is not readily available or constrained due to local resources.It is now evident that both in the USA and internationally, risks of proceeding with living donor transplant with its potential impact on patients and healthcare resources need to be balanced with the risks of continued dialysis where challenges to social distancing, increased demand and worse outcomes related to COVID-19 exposure are increasingly understood.et al show that liver transplants from living and deceased donors were 49%, and 9% lower compared with historical trends, respectively.Although there was no appreciable difference between 2019 and 2020 in the number of living donor liver cases performed, subsequent surges of the ongoing COVID-19 pandemic may pose a threat to the volume of living liver donor transplants given local conditions. From 15 March 2020 to 31 August 2020, Strauss We posit that there are indeed harms to delaying living liver donor transplants because this subset of patients does not have options for sustaining survival other than transplantation and, as such, represent a patient population with a high level of medical urgency. From an ethical perspective, the rule of rescue supports continuance of these procedures as it suggests that it is an imperative to advocate on behalf of an individual patients’ need who face a clear fatal outcome, regardless of expense.In light of the ethical analysis above, we propose some modest conclusions intended to be used as resources of guidance. These recommendations apply to transplant hospitals experiencing contingency standards of care and may not be applicable in conditions guided by crisis standard of care.Living donor transplants represent an important option for individuals with end-stage liver and kidney disease and should not be suspended uniformly when the aim is to provide contingency standards of care. Decisions to delay living donation may be appropriate given local conditions. However, these decisions should have on-going evaluation, be communicated transparently and include timely dialogue with patients.Exposure risk to SARS-CoV-2 should be balanced with other risks, such as exposure risks at dialysis centres. Because many of these risks are not quantifiable and variable given local case rates, donors and recipients should be engaged in the process of documented shared decision-making, in order to understand their perspectives of what constitutes risk as well as to come to a mutual appreciation of the social context of donation which informs the willingness to undertake these risks.Transplant hospitals should strive to maintain a critical transplant workforce in order to avoid diverting this unique expertise in a manner, which could negatively impact patient preparedness for transplant. Under crisis standards, it is understandable that every available resource is optimised to provide prioritised care. However, under contingency standards, transplant centres continue to provide equivalent services and, as such, should do so with an optimised workforce familiar with the complexities of waitlist management, transplant organ offers and post-transplant management.As the understanding of COVID-19 outcomes in immunosuppressed patients continues to evolve,Many transplant hospitals designate living donor transplantation as an elective procedure, which makes these operations susceptible to temporary suspensions in favour of procedures categorised as emergent or urgent. Although CMS designates transplantation at a high level of medical urgency (tier 3b), the elective nature of living donor transplantation reflects the general agreement that a living donor undergo’s surgery mainly to benefit others and should so without pressure or urgency. It is understandable that under crisis standards, living donor transplant procedures should be delayed in favour of allocating crucial resources to the population-at-large. Under contingency standards, which offers functionally equivalent care delivery, additional considerations applicable to a specific environment are necessary. For example, to assure standards of contingency during an outbreak of COVID-19 hospitalisations, additional protocols to minimise infectious transmission and to assure an adequate transplant workforce are key elements that need to be discussed with patients. Given the uncertainty surrounding the risks and outcomes associated with hospital exposure to SARS-CoV-2, advocating for continuance of these procedures under contingency standards of care should be undertaken by engaging donor/recipient pairs in a process of shared decision-making and within a framework of comprehensive informed consent. Although this proposed ethical framework was developed in the context of the COVID-19 pandemic, it may also be applicable in situations where local healthcare resources are strained, where continency standards are in effect, and where critical decisions regarding healthcare priorities need to be made.


This cohort study compares the use and outcomes of high-sensitivity cardiac troponin assay vs conventional troponin assay in transgender adults. Medical transitioning includes gender-affirming hormone therapy.5 Exposure to hormone therapy affects levels of cardiac biomarkers.6 There is no established cardiac troponin diagnostic threshold for transgender individuals. This study aimed to determine the role of implementing sex-specific troponin thresholds in the diagnosis and management of suspected acute coronary syndrome (ACS) in transgender patients.Diagnosing acute myocardial infarction (AMI) requires the detection of cardiac troponin above the 99th percentile upper reference limit.STROBE reporting guideline.This population-based cohort study included Kaiser Permanente Southern California (KPSC) members (aged ≥18 years) presenting with suspected ACS between 2007 and 2022. The KPSC Institutional Review Board approved the study and waived the informed consent requirement because the study presented minimal risks. We followed the 2 Patients were followed up for 30 days for outcomes.Transgender patients were identified through review of an internal registry, hormonal treatment history, sex assigned at birth, and medical records. Patients never treated with hormonal therapy were excluded. In 2021, the KPSC health system implemented a high-sensitivity cardiac troponin I (hs-cTnI) assay  with sex-specific diagnostic thresholds. Prior to 2021, a conventional troponin assay with a single threshold was used. Troponin levels on presentation were identified, and cause of troponin elevation was adjudicated by physicians using the universal definition of myocardial infarction (MI).2 test. Two-sided P < .05 defined statistical significance. Statistical analyses were conducted with Stata/MP 17.0 (StataCorp LLC).Outcomes before and after the hs-cTnI assay implementation were compared using the χAmong the 1108 patients included, 661 (59.7%) were transgender women and 447 (40.3%) were transgender men, with a median (IQR) age of 33 (25-47) years. Overall, patients self-identified as Asian (77 [6.9%]), Black (108 [9.8%]), Hispanic (360 [32.5%]), or White (524 [47.3%]). Two hundred sixty-two patients (23.6%) were evaluated after implementation of the hs-cTnI assay with sex-specific thresholds; all other patients (846 [76.4%]) were evaluated using a conventional troponin assay with a single threshold. With the conventional troponin assay, 24 individuals (2.8%) had abnormal troponin levels . With thThree patients (1.1%), who would be reclassified if their sex assigned at birth were used for the threshold, had type 2 MI or myocardial injury. No patients with type 1 MI would have been reclassified. No significant differences in clinical outcomes at 30 days were observed before and after implementation of sex-specific troponin diagnostic thresholds.In this cohort study of transgender patients, implementing sex-specific troponin thresholds for diagnosing AMI did not significantly affect outcomes. Use of sex-specific thresholds aligned with affirmed gender identity was not associated with significant reclassification. None of the few reclassified patients had myocardial injury due to type 1 MI. Patients presenting with ST-segment elevation MI and type 1 non–ST-segment elevation MI had troponin levels that were much higher than the diagnostic thresholds and were appropriately identified regardless of the threshold used.Study limitations were short follow-up time, small sample size, and the observational design. The findings suggest that troponin thresholds in accordance with the patient’s affirmed gender identity may be used in transgender individuals. Doing so may reduce confusion and distress for both patients and clinicians and help patients receive truly gender-affirming care.


During biological aging, significant metabolic dysregulation in the central nervous system may lead to cognitive decline and neurodegeneration. However, the metabolomics of the aging process in cerebrospinal fluid (CSF) has not been thoroughly explored.In this cohort study of CSF metabolomics using liquid chromatography-mass spectrometry (LC–MS), fasting CSF samples collected from 92 cognitively unimpaired adults aged 20–87 years without obesity or diabetes were analyzed.We identified 37 metabolites in these CSF samples with significant positive correlations with aging, including cysteine, pantothenic acid, 5-hydroxyindoleacetic acid (5-HIAA), aspartic acid, and glutamate; and two metabolites with negative correlations, asparagine and glycerophosphocholine. The combined alterations of asparagine, cysteine, glycerophosphocholine, pantothenic acid, sucrose, and 5-HIAA showed a superior correlation with aging (AUC = 0.982). These age-correlated changes in CSF metabolites might reflect blood–brain barrier breakdown, neuroinflammation, and mitochondrial dysfunction in the aging brain. We also found sex differences in CSF metabolites with higher levels of taurine and 5-HIAA in women using propensity-matched comparison.Our LC–MS metabolomics of the aging process in a Taiwanese population revealed several significantly altered CSF metabolites during aging and between the sexes. These metabolic alterations in CSF might provide clues for healthy brain aging and deserve further exploration.The online version contains supplementary material available at 10.1186/s12877-023-03939-6. World Population Ageing Report published by United Nations, the size of the population aged above 65 will increase by 120% from 0.7 billion in 2019 to 1.5 billion in 2050, and 64% of the aged population in 2050 will be living in Asia [Over the past 60 years, improvements in healthcare and a booming economy have led a marked increase in the average human lifespan by almost 23 years . AccordiDuring brain aging, dysregulated bioenergetics, neuroplasticity, and neuroinflammation contribute to the risk of cognitive decline and neurodegenerative disorders . Age-relAging-related degradation of molecular and cellular processes contribute to genome, proteome, and lipidome instability, and the minor changes of these upstream molecules can cause significant metabolite alterations . BecauseCerebrospinal fluid (CSF) is an appropriate biospecimen for analyzing the aging of the central nervous system (CNS), because CSF interacts closely with CNS tissue and its composition can reflect brain-specific metabolite alterations during aging. CSF exchanges metabolites between the cerebral and systemic circulation; however, the specialized tight junctions of the BBB limit transcellular transport in the CNS . AlthougIn the current metabolomic study, the LC–MS metabolomic profiles of CSF samples collected from cognitively healthy patients were analyzed to examine the metabolomic alterations during the aging process and between different sex. This clinical study was deposited in the Clinical Trials Registry  and approved by the Institutional Ethical Review Board . We have explicitly explained the study protocol to every participant before enrollment into the study, and written informed consent was obtained after explanation.We enrolled adult participants who were cognitively healthy  and were receiving optional spinal anesthesia for elective surgery at Linkou Chang Gung Memorial Hospital, a tertiary medical center in Northern Taiwan. The study participants were divided into three groups by age: young (age 20–39 years), middle-aged (40–59 years), and old (aged ≥ 60 years). The three-age group classification has been applied in previous LC–MS metabolomic study of human aging . Patient2 or a fasting blood glucose > 126 mg/dL from the analysis because these patients were considered obese or diabetic, respectively, according to diagnostic criteria [Cognitive evaluation was based on preoperative assessment and the exclusion of neurological or psychiatric diseases; thus, mild cognitive impairment might have been overlooked. After the initial evaluation, we excluded eight participants with a body mass index (BMI) > 30 kg/mcriteria . The finThe CSF collection procedures were largely the same as that described in our previous NMR metabolomic study . After oThe CSF samples (50 μL) were mixed with cooled methanol (200 μL) to precipitate proteins. After centrifugation at 12,000 × g for 15 min, the supernatant was transferred to nitrogen gas for drying. The residue was suspended in 200 μL of 50% acetonitrile for LC–MS analysis.Liquid chromatographic separation was conducted on an ACQUITY UPLC BEH Amide column  using an ACQUITY TM Ultra Performance Liquid Chromatography (UPLC) system (Waters Corp.). The column was maintained at 45 °C, and the flow rate was 0.4 mL/min. The mobile phase consisted of 0.1% formic acid in water (phase A) and acetonitrile containing 0.1% formic acid (phase B). Mass spectrometry was performed on a Waters Q Tof–MS  operated in ESI positive and negative ion modes. The scan range was 50–1000 m/z. The desolvation gas flow rate was 800 L/hr at 500 °C. The source-cone voltage was set to 25 V. The capillary voltage was 2.5 kV in positive mode and 2 kV in negative mode. The lock mass was leucine encephalin (m/z: 120.0813 and 556.2771 for positive mode and m/z: 236.1035 and 554.2615 for negative mode).Finally, 162 CSF compounds were identified by LC–MS using an in-house library of CSF samples. The in-house library of CSF samples was set up by standard metabolite annotation or MS/MS fragment verification. Metabolite annotation was performed using accurate mass, retention time, and MS/MS criteria. Raw data can be obtained from the corresponding author. Of the 162 compounds, 37 CSF metabolites could be characterized using the CSF metabolome database of the Human Metabolome Database (HMDB) with high confidence . Further2 tests (for two-groups) and analysis of variance (ANOVA) (for multiple groups). The between-group differences of a specific metabolite were compared using the OPLS-DA coefficients of the LC–MS signals, and the variables in the OPLS-DA score plots were compared using goodness of fit .The recorded data are expressed as means ± SD for continuous variables and percentages for qualitative variables (sex and diseases). The statistical analyses in our study were based on the acquired LC–MS signal integration of each metabolite, and data were compared using Student’s t-test or χp value less than 0.05 was defined as statistically significant.In this study, CSF metabolite profiling of cognitively healthy adults of different age groups was the primary outcome. The aim of this analysis was to identify CSF metabolites that could discriminate between the young and old age groups, and then calculate the correlation between metabolite abundance and aging using a regression model. Metabolites with significant discrimination between the old and young groups were selected to construct metabolite combinations; those combinations with lower AIC values, higher AUC values, or higher odds ratios (ORs) were selected to evaluate their discrimination of these two age groups. Between-group comparisons were adjusted for sex, BMI, serum creatinine level, and medical history of hypertension since these variables had significant between-group differences in the demographic comparisons. As the aging process might differ between the sexes, we calculated the correlation of each metabolite with aging in male and female adults. The secondary outcome was metabolite comparison between different sex. We made a 1:1 propensity-score matched comparison between male and female adults by matching participants by age, BMI, serum creatinine level, and hypertension, and then compared their differences in metabolite abundance between male and female adults. All statistical analyses were performed on SAS software , and a two-sided n=34), middle-aged (n=31), and old (n=27) groups. The study protocol is shown in Fig. Our final cohort included 92 patients, which were divided into the following three age groups: young .The OPLS-DA score plots of the LC–MS signals between age groups are shown in Fig. p < 0.05). The old age group had lower levels of asparagine and glycerophosphocholine than the young group. These age-related metabolite alterations were visualized in a boxplot for metabolite comparison between young, middle-aged, and old age groups according to LC–MS signal integrations , whereas α-ketoglutarate (α-KG), glutamine, serine, glycerophosphocholine, and asparagine were negatively correlated with age . Figure 2 transformed metabolite abundance and their specific correlations within the three age groups. To identify aging-correlated biomarkers in the cerebral circulation, we constructed metabolite combinations using CSF metabolites with significant discrimination between young and old patients. We then compared the AIC and AUC values and adjusted ORs of these combinations for discriminating between the young and old age groups utilizing stepwise ANOVA and multivariate analysis, and the results are shown in Table The correlations of the log2 transformed metabolite abundance and their correlations with aging within the male and female groups is shown in Fig. The aging process might differ between males and females; therefore, we divided our cohort according to sex, and the demographic comparison is shown in Table p < 0.05), and the boxplots of the propensity-matched between-sex comparisons are shown in Fig. To further compare the differences in CSF metabolites between males and females, we utilized propensity score-matched comparison by 1:1 matching according to age, BMI, serum creatinine, and hypertension to eliminate these confounders. After propensity score matching, no obvious between-group differences were observed Table . BetweenThe metabolites that are altered during aging are mainly related to amino acids, lipids, neurotransmitters, and energy metabolism. Enrichment analysis of these altered metabolites showed significantly higher enrichment ratios for aminoacyl-tRNA biosynthesis, glutamine and glutamate metabolism, butanoate metabolism, and pantothenate and CoA biosynthesis. Pathway analysis of these age-correlated metabolites showed higher correlations with alanine, aspartate, and glutamate metabolism; arginine biosynthesis; glutamine and glutamate metabolism; and aminoacyl-tRNA biosynthesis. The involved metabolic pathways and their profiled metabolic changes during brain aging process of these age-correlated metabolites are depicted in Fig. This prospective cohort study intended to profile the metabolomic signature in CSF during aging in cognitively unimpaired Taiwanese adults using LC–MS. Metabolomic analysis of fasting CSF samples showed significantly higher levels of pantothenic acid, 5-HIAA, sucrose, glutamate, and 2-HG and lower levels of asparagine and glycerophosphocholine in the old age group than in the young group. The combination of asparagine, cysteine, glycerophosphocholine, pantothenic acid, sucrose, and 5-HIAA was strongly correlated with aging, with an AUC of 0.982 to discriminate the old age group from the young group. We also found higher levels of taurine, hypoxanthine, and 5-HIAA in CSF samples from females than in CSF samples from males using a 1:1 propensity-score matched comparison. These aging-related CSF metabolites indicate decreased lipid, amino acid, neurotransmitter, and energy metabolism, suggesting possible BBB breakdown, neuroinflammation, and mitochondrial dysfunction in aged brain circulation.This LC-MS metabolomic analysis of healthy aging is an extension of our previous NMR analysis of aging metabolomics. The two most common metabolomic analytic methods are NMR spectroscopy and mass spectrometry-based metabolomics. While NMR spectroscopy can identify core metabolites in key metabolic pathways, mass spectrometry-based metabolomics can identify low abundance metabolites with wide detection range and excellent sensitivity . In our Pantothenic acid, also known as vitamin B5, is the most altered metabolites in this LC–MS analysis. Pantothenic acid is an essential trace nutrient important for the synthesis of coenzyme A, which plays an important role in the tricarboxylic acid (TCA) cycle, fatty acid metabolism, acetylcholine, and myelin synthesis . A recen5-HIAA is a major metabolite of serotonin. A previous CSF metabolomics study of subjects with AD and mild cognitive impairment found that these patients had elevated levels of methionine and 5-HIAA . GlutamaCysteine, a non-essential amino acid, is metabolized from the methionine and can be converted to glutathione, an important antioxidant and free radical scavenger . CysteinPseudouridine, also known as the ‘fifth nucleotide’ of RNA, is an isomer of the nucleoside uridine and plays an important role in the metabolism of purine nucleosides, muscle amino acids, and organic acids . RNA pseC. elegans by binding to and inhibiting ATP synthase, thereby decreasing mitochondrial respiration and mTOR signaling [C. elegans by inhibiting ATP synthase and TOR signaling and suppressed chronic inflammation in female mice [2-HG is structurally similar to α-KG and is associated with tumorigenesis and neurological dysfunction . 2-HG exignaling . Mutatioignaling . In our ignaling . A recenignaling . Besidesale mice . DecreasAsparagine, a non-essential amino acid, is essential for the brain development and function, and an asparagine synthetase deficiency can lead to congenital microcephaly and neuronal damage . AsparagGlycerophosphocholine, a glycerophospholipid with choline as a headgroup, is the main component of biological membranes and is a reservoir for second messengers. Alteration of the glycerophospholipid composition of neural membranes may change neural membrane permeability and is correlated with neurodegenerative diseases . A largeThe altered levels of asparagine, cysteine, pantothenic acid, sucrose, 5-HIAA, and glycerophosphocholine in the CSF samples of the older group in our study suggest decreased lipid, amino acid, neurotransmitter, and energy metabolism in the aged brain. A previous Swedish study of CSF metabolomics found significant positive associations between age and acetylcarnitine, glutarylcarnitine, hippurate, 5-hydroxytryptophan, isoleucine, ketoleucine, methionine, and pipecolate and negative correlations between age and methylthioadenosine and 3-methy-ladenine . A recenExamining samples from healthy adults is of paramount importance as it could help us understand the healthy aging process and how aging trajectories differ between the sexes, thus enabling us to identify pathogenic phenomena and targeted therapeutics for neurodegenerative diseases. Exploring the sex differences between male and female adults was our secondary outcome. Previous plasma metabolomics studies exploring sex differences in aging found that the significant differences between men and women could be attributed to five metabolic pathways: primary bile acid biosynthesis, lysine degradation, fatty acid biosynthesis, linoleic acid metabolism, and the pentose phosphate pathway . AnotherOur CSF metabolomics analysis of cognitively healthy adults revealed decreased phospholipid, amino acid, neurotransmitter, and energy metabolism during aging, suggesting aging-related metabolic changes in BBB breakdown, neuroinflammation, and mitochondrial dysfunction, which is in line with previous studies on brain aging . With beTo the best of our knowledge, this CSF metabolomic study is the first to profile the LC–MS metabolomic signature of healthy brain aging in the Asian population. Compared to previous CSF metabolomics studies, our metabolomic cohort has a homogeneous ethnicity, adequate patient number, and case information . Our CSFAlthough this study was metabolically well characterized, it had some limitations. First, we only collected CSF samples once from the study participants, so we could not compare matched metabolomic information from other samples, such as plasma or urine. In fact, it is not yet known whether the changes in the analyzed metabolites represent neutral changes with age, sudden dynamic stress response, or merely individual variations.  ConsiderIn this cross-sectional cohort study of LC–MS metabolomic profiling of human CSF samples from cognitively healthy adults in the Taiwanese population, we presented novel insights into metabolic dysregulation in cerebral circulation during aging. These identified CSF metabolite changes in aging process are involved in metabolism of lipid, amino acid, neurotransmitter, and mitochondrial respiration. The profiled aging metabolomic changes might imply defected cellular signalling, BBB breakdown, neuroinflammation, and mitochondrial dysfunction in the aging cerebral circulation. Furthermore, a combined CSF alteration of asparagine, cysteine, glycerophosphocholine, pantothenic acid, sucrose, and 5-HIAA displayed a superior correlation with the aging process, which may provide clues for healthy brain aging and deserve further investigation for causal relationships.Additional file 1:Supplementary Materials: Table S1. Comparison of CSF metabolite abundance in different age group.Additional file 2:Figure S1. Boxplot for metabolite comparison between young, middle, and old age group.Additional file 3:Figure S2. Metabolite heatmaps in CSF samples. (A) Young versus old group, (B) Young versus middle versus old group.


The objective of this systematic review is to identify how reporting of micro-invasive glaucoma surgery (MIGS) procedure complications are reported in randomised controlled trials (RCTs) and the quality of this reporting compared to the CONSORT extension for harms. RCTs evaluating MIGS procedures were identified from a database of systematic reviews and from recent literature. Trials were evaluated in comparison to the CONSORT extension for harms to quantify the quality of harms reporting. Simple descriptive statistics were calculated for the CONSORT checklist. 21 trials were identified as eligible for inclusion, 14 were evaluating iStent, one Trabectome, three Hydrus, one Cypass, one Preseflo MicroShunt and one Excimer laser trabeculotomy. The average number of CONSORT for Harms checklist items fulfilled by the studies was 10 out of 16. No studies used a validated instrument to report severity of harms and only 4 had a list or definition of adverse events. An analysis of harm was conducted by 19 of 21 studies (90%). Appropriate metrics were used for reporting rates of adverse events in 19 of 21 studies but in only 4 studies was there an attempt to give these adverse events a grade of seriousness. In conclusion, most studies evaluating MIGS procedures do make an effort to acknowledge harms data, however this is not done uniformly well or in the same manner. A validated instrument to report severity and a standard list of complications for MIGS surgery would go a long way to helping this. Over the past decade there has been a paradigm shift in the world of glaucoma surgery from traditional procedures such as the trabeculectomy and tube drainage devices to a wide range of new techniques and devices which purport to be able to lower intraocular pressure (IOP) in a less invasive manner. These are collectively called micro-invasive glaucoma surgery (MIGS), although there is no widely accepted definition of what can and cannot be referred to by this term. One of the key tenets of some of the novel procedures is that although they may not lower IOP as much as traditional surgery, they are safer , particuIn 2019, the World Glaucoma Association published consensus guidelines on the design and reporting of glaucoma surgical trials and included in this, guidelines on the reporting of complications . This haAdequate reporting and quantification of severity of complications is an important consideration when evaluating surgical innovations. Sii et al. highlighted deficiencies in the reporting of complications in glaucoma surgical trials . This reIn this study, we identified how complications were reported and the quality of the reporting in MIGS trials.We identified systematic reviews and randomised controlled trials (RCTs) on surgical interventions for glaucoma. The protocol for this review has been registered in the online database PROSPERO (CRD42021278766).The Cochrane Eyes and Vision United States Satellite maintains a database of Cochrane and non-Cochrane systematic reviews and meta-analyses in vision research and eye care. The full search strategy for this database has been published elsewhere . We compThe population of interest was adult patients with glaucoma of any type, with or without co-existing cataract. As intervention, we considered any novel glaucoma surgery, including MIGS procedures performed for any reason, either alone or in combination with cataract surgery. We excluded studies evaluating outcomes of traditional glaucoma surgery , interventions for congenital glaucoma, and laser therapy.As comparator we included any control or alternative intervention.For each trial identified data on the reporting of complications was extracted by one investigator and checked by a second investigator against the CONSORT extension for harms criteria . We did The CONSORT extension for harms contains ten recommendations about reporting harms-related issues. Some of these are quite broad and so were subdivided to enhance the quality of data collection and its ease Table  8]. Eac. Eac8]. The PRISMA flowchart Fig.  and a liA total of 21 trials were identified as eligible for inclusion. Of the 21 included studies, 14 were evaluating iStent, one Trabectome, three Hydrus, one Cypass, one Preseflo MicroShunt and one Excimer laser trabeculotomy outcomes . No studies used a validated instrument to report severity, only 4 had a list or definition of adverse events and only 4 differentiated between expected and unexpected events (Fig. With the introduction of novel surgical techniques an evaluation of efficacy and harms is essential for surgeons and patients to be able to compare and choose among different procedures.As we think back to the key principles of medicine, the concept of ‘primum non nocere’ or ‘first do no harm’ is a cornerstone of modern practice. We must therefore acknowledge that reporting of surgical complications is one of the most important metrics when evaluating a new surgical technique or device for use. The CONSORT extension for harms was designed to help reporting this important domain in a thorough and structured manner. From our review, we can see that most studies are attempting to consider harms related data as part of their approach. It is apparent however that some factors are not present which would aid this. No studies used a validated instrument to report severity and very few used a list or definitions. We feel that these would assist greatly in the study of harms related data in glaucoma surgical trials of MIGS surgery. Stringa et al. have recently published a list of complications of glaucoma surgery . This coIt is also important to acknowledge that RCTs may not be able to identify uncommon complications due to their sample size, with very large RCTs powered to detect these complications being too large and expensive to run. Registries where surgeons report clinical outcomes and complications or real world data are better suited to detect less frequent complications of surgical interventions.Our study does have some limitations, there are not yet many RCTs evaluating harms in MIGS surgery and so we could only include a relatively small number of studies. Many MIGS techniques are also quite new and so longer term data regarding safety has yet to be established. The strengths however are the systematic search and also the use of the CONSORT extension for harms which is a well established method of reporting harms in RCTs. This study complements the recent overview on MIGS devices and highlights that we have no robust evidence to be able to compare effectiveness and safety among different devices .


Inflammasomes and innate immune cells have been shown to contribute to liver injury, thereby activating Kupffer cells, which release several cytokines, including IL-6, IL-1ß, and TNFα. Augmenter of liver regeneration (ALR) is a hepatotropic co-mitogen that was found to have anti-oxidative and anti-apoptotic properties and to attenuate experimental non-alcoholic fatty liver disease (NAFLD) and cholestasis. Additionally, hepatic ALR expression is diminished in patients with NAFLD or cholestasis, but less is known about the mechanisms of its regulation under these conditions. Therefore, we aimed to investigate the role of IL-1ß in ALR expression and to elucidate the molecular mechanism of this regulation in vitro. We found that ALR promoter activity and mRNA and protein expression were reduced upon treatment with IL-1ß. Early growth response protein-1 (Egr-1), an ALR inducer, was induced by IL-1ß but could not activate ALR expression, which may be attributed to reduced Egr-1 binding to the ALR promoter. The expression and nuclear localization of hepatocyte nuclear factor 4 α (HNF4α), another ALR-inducing transcription factor, was reduced by IL-1ß. Interestingly, c-Jun, a potential regulator of ALR and HNF4α, showed increased nuclear phosphorylation levels upon IL-1ß treatment but did not change the expression of ALR or HNF4α. In conclusion, this study offers evidence regarding the regulation of anti-apoptotic and anti-oxidative ALR by IL-1ß through reduced Egr-1 promoter binding and diminished HNF4α expression independent of c-Jun activation. Low ALR tissue levels in NAFLD and cholestatic liver injury may be caused by IL-1ß and contribute to disease progression. The liver plays a critical role in numerous physiological processes, including synthesis of proteins, hormones, and bile acids; catabolism of xenobiotics; and metabolism of carbohydrates, cholesterol, and lipids. Hepatocytes are the liver cell type that fulfil the majority of these functions, thereby reflecting their important role in maintaining metabolic homeostasis . BecauseLiver diseases, such as non-alcoholic steatohepatitis (NASH)  and cholAugmenter of liver regeneration (ALR), a member of the ALR/Erv1 protein family, has been shown to augment the process of liver regeneration . ALR is To analyze the activity of the ALR promoter upon IL-1ß treatment, HepG2 cells were transfected with a full-length ALR promoter construct (−733/+527)  and subsgrowth factor erv1-like (GFER) gene (coding for ALR) promoter encompasses binding sites for several transcription factors, including HNF4α [The ng HNF4α , which hng HNF4α . TherefoEgr-1, a zinc finger transcription factor, induces ALR expression by binding to its response element within the ALR promoter . IL-1ß hIt has been reported that c-Jun binds to the AP-1/AP-4 binding site within the ALR promoter and consequently represses ALR promoter activity . TherefoIn this study, we present evidence for the negative regulation of ALR expression by IL-1ß. ALR mRNA and protein expression were reduced upon IL-1ß treatment, which is partially mediated by diminished HNF4α expression and HNF4α nuclear localization and also possibly by reduced binding of Egr-1 to the ALR promoter. On the contrary, the transcription factors c-Jun and AP-4 did not reduce ALR expression per se and did not seem to play a role in the ALR reduction caused by IL-1ß.Augmenter of liver regeneration (ALR) is an anti-apoptotic and ER stress-reducing protein ,22. FurtHowever, little is known about the regulation of ALR expression by Kupffer cell-released IL-1ß. We assessed the regulation of ALR by IL-1ß using luciferase assays, qRT-PCR, and Western blot techniques and found that ALR expression and promoter activity are reduced by IL-1ß. It has previously been reported that IL-1ß can induce AP-1 expression ,45 and tFurthermore, it was shown that IL-1ß decreases HNF4α mRNA and protein expression while increasing phosphorylated c-Jun levels in human liver cells ,48. Addiγ [Expression of Egr-1, an inducer of ALR expression , is enhaγ  and typeγ  promoterTo shed more light on the role of ALR in liver disease development, studies have been performed in which ALR was silenced and the outcome of its absence was monitored . BrieflyInflammation and oxidative stress are also observed in another common liver disease, cholestasis. Cholestasis is a pathological syndrome caused by impaired or disrupted bile flow from the liver, resulting in hepatic apoptosis , oxidatiInflammation in NASH and cholestasis is characterized not only by enhanced levels of IL-1ß but also by increased release of IL-6 and TNFα ,55. IL-6While most researchers have focused on the role of ALR in disease development by performing loss and gain of function models, less is known about the molecular mechanisms of ALR regulation. Here, we present evidence that IL-1ß reduces ALR mRNA and protein expression through diminished HNF4α expression and decreased Egr-1 binding to the ALR promoter independent of c-Jun activation. ALR was reported to have anti-apoptotic and anti-oxidative properties, resulting in less oxidative stress and presumably less induction of pro-inflammatory cytokines. Even though ALR expression is induced by oxidative stress, excessive levels of IL-1ß might impede proper ALR expression for cell protection and regeneration. Low ALR expression levels have been reported in liver tissue from patients with NASH and cholestasis, and the observed high IL-1ß levels in these patients may be causative. Further studies investigating the impact of IL-1ß on ALR’s hepatoprotective capacity, expression of ALR under inflammatory conditions, and correlations between IL-1ß and ALR levels in patients would be of interest.Cell culture and treatments: Cells from the human hepatoma cell line HepG2, obtained from the American Type Culture Collection , and Huh-7 cells (ECACC 01042712), obtained from the European Collection of Authenticated Cell Cultures (ECACC) , were grown at 37 °C, 5% CO2 in DMEM  supplemented with penicillin (100 units/mL), streptomycin (10 μg/mL), and 10% fetal calf serum . Cells were seeded at a density of 4 × 104 cells/cm2 and cultured for 24 h, followed by 18–24 h starvation (FCS-free conditions) as indicated and treated with the indicated concentrations of IL-1ß . Primary human hepatocytes (PHH) were isolated and maintained in culture, as described earlier [ earlier ,60, and Reporter gene assays: HepG2 cells were cultured at a density of 5 × 104 cells/cm2 and then transfected using Lipofectamine® 3000  with 200 ng of human ALR promoter construct (−733/+527) [2-basic served as a negative control. Each experiment was repeated at least three times.33/+527) . AdditioTransient transfection with HNF4α and c-Jun and silencing c-Jun expression: HepG2 cells were transfected using Lipofectamine® 3000  with 2.5 µg of pcDNA3.1-HNF4α , pcDNA3.1-c-Jun-V5  [ty, USA) , or 30 pRNA isolation, reverse transcription, and qRT-PCR: Total RNA was isolated using an RNeasy Mini Kit . One microgram of total RNA was reverse-transcribed using a Reverse Transcription System from Qiagen . Transcription levels of ALR, HNF4α, Egr-1, SP1, c-Jun, AP-4, and HPRT were quantified using real-time PCR technology . Primer pairs were designed as reported earlier . PCR reaction products were verified using sequence analysis, and each quantitative PCR was performed in triplicate. The primers used for qRT-PCR are listed in lier see  or with SDS-PAGE and immunoblotting: Total proteins were isolated, prepared, and subjected to Western blot analysis. Briefly, 30 μg per lane was separated by 12% SDS-PAGE , and proteins were transferred onto PVDF membranes , incubated with specific antibodies, and developed using an enhanced chemiluminescence reagent . Anti-ALR polyclonal antibody was “custom made” against 15 kDa ALR (short form ALR) prepared by Davids Biotechnology . The following antibodies were used: anti-ß-actin (#4970); anti-HNF4α (#3113) obtained from Cell Signaling ; anti-HDAC (#05–100) purchased from Merck ; and anti-Egr1 (#189), and anti-phospho-c-Jun (#822) purchased from Santa Cruz Biotechnology . Secondary goat HPR-conjugated antibodies (anti-rabbit #P0448 and anti-mouse #P0447) were obtained from Dako .Electrophoretic mobility shift assay (EMSA): EMSA was performed as published earlier [ttgcccccgcccaggta-3′ and Egr-1-ΔSP1-Rev: 5′-tacctgggcgggggcaaggacagg-3′. The biotin-labeled DNA was then detected using an enhanced chemiluminescence reagent . Competition experiments were performed using a 200-fold excess of the same unlabeled binding site 20 min prior to electrophoresis using 6% DNA retardation gels  at room temperature. earlier . BrieflyStatistical analysis: All data are presented as mean +/− standard deviation or standard error of the mean as indicated. Data were compared between groups using Student’s t-test or ANOVA with post-hoc Bonferroni correction where appropriate. Values of p < 0.05 were considered significant .


The prognosis of patients with leptomeningeal metastasis (LM) remains poor. Circulating tumour DNA (ctDNA) has been proven to be abundantly present in cerebrospinal fluid (CSF); hence, its clinical implication as a biomarker needs to be further verified.We conducted a retrospective study of 35 lung adenocarcinoma (LUAD) patients with LM, and matched CSF and plasma samples were collected from all patients. All paired samples underwent next-generation sequencing (NGS) of 139 lung cancer-associated genes. The clinical characteristics and genetic profiling of LM were analysed in association with survival prognosis.P = 0.003), a higher median mutation count (P < 0.0001), a higher frequency of driver mutations (P < 0.01), and more copy number variation (CNV) alterations (P < 0.001) than plasma. The mutation frequencies of the EGFR, TP53, CDKN2A, MYC and CDKN2B genes were easier to detect in CSF than in LUAD tissue (P < 0.05), possibly reflecting the underlying mechanism of LM metastasis. CSF ctDNA is helpful for analysing the mechanism of EGFR-TKI resistance. In cohort 1, which comprised patients who received 1/2 EGFR-TKIs before the diagnosis of LM, TP53 and CDKN2A were the most common EGFR-independent resistant mutations. In cohort 2, comprising those who progressed after osimertinib and developed LM, 7 patients (43.75%) had EGFR CNV detected in CSF but not plasma. Furthermore, patient characteristics and various genes were included for interactive survival analysis. Patients with EGFR-mutated LUAD (P = 0.042) had a higher median OS, and CSF ctDNA mutation with TERT (P = 0.013) indicated a lower median OS. Last, we reported an LM case in which CSF ctDNA dynamic changes were well correlated with clinical treatment.LM showed genetic heterogeneity, in which CSF had a higher detection rate of ctDNA (CSF ctDNA could provide a more comprehensive genetic landscape of LM, indicating the potential metastasis-related and EGFR-TKI resistance mechanisms of LM patients. In addition, genotyping of CSF combined with clinical outcomes can predict the prognosis of LUAD patients with LM.The online version contains supplementary material available at 10.1007/s11060-023-04471-8. EGFR-mutant patients is 9.4% [EGFR), targeted therapy has begun a new chapter based on an actionable molecular alteration in lung cancers [Leptomeningeal metastasis (LM) is a severe complication associated with metastatic solid tumours that is caused by neoplastic cells transferring to the pia mater, arachnoid, and subarachnoid space . The inc is 9.4% . Since t cancers . Unfortu cancers .It is essential to characterize genomic diversity between primary and metastatic cancers. Nevertheless, tissue biopsies are invasive procedures, and some locations are not easy to access, such as the bones and brain . HoweverDue to the existence of the blood‒brain barrier (BBB), genotyping of the primary tumour and plasma does not represent the genetic alterations of cerebrospinal fluid (CSF) in LM. Currently, CSF is considered a crucial liquid biopsy medium for LM that provides an accessible and less invasive method to acquire information on genomic alterations . CSF ctDIn this retrospective cohort study, we collected data from LUAD patients with LM who were enrolled in the Department of Neurology, The Second Hospital of Hebei Medical University , from December 2015 to December 2022. The inclusion criteria were as follows: (1) > 18 and ≤ 75 years old; (2) diagnosis of LM according to the European Association of Neuro-Oncology-European Society for Medical Oncology (EANO-ESMO) clinical practice guidelines [To study clinical outcomes, overall survival (OS) was measured. OS was defined as the time from LM to death or last follow-up.Freshly frozen CSF, and whole blood samples were collected for genomic profiling. Total DNA from freshly frozen CSF and plasma was extracted by QIAamp Circulating Nucleic Acid Kit .The Illumina libraries were carried out with KAPA Hyper Prep Kit  according to the manufacturer’s protocol. Targeted panel sequencing was performed by SeqCap EZ System , and obtained by the Geneseeq Prime™ panel  which was covering 139 cancer-associated genes. On HiSeq X10 sequencing system , enriched libraries were sequenced with 150 bp pair-end reads.P values were 2-sided and were considered statistically significant at P < 0.05. Multivariate analysis was performed using Cox proportional hazard regression with inclusion of variables significant on univariate regression.Survival analysis was performed using Kaplan‒Meier method with log-rank P values and 95% confidence intervals (CIs) reported. All Associations between two variables were analyzed using Fisher’s exact, Chi-square tests or Wilcoxon test. Graph Pad Prism version 9.0, SPSS version 25.0 and R version 4.1.2 software used for statistical analyses and making graphs.EGFR mutations were found in 26 patients (74.29%) at the initial diagnosis of LUAD. Among them, 13 patients (37.14%) were diagnosed with LM and BMs simultaneously, and 23 patients (65.71%) had extracranial metastases. Most of the patients suffered from neurologic symptoms prior to LM diagnosis. The most common clinical manifestations were cerebral symptoms (97.14%), such as headache and dizziness. Posterior fossa symptoms, including various cranial neuropathies (51.43%) and spinal cord/root symptoms, such as motor and sensory dysfunction (13%), were also in evidence. Thirty-three patients (94.29%) received targeted therapy and other therapy combinations (angiogenesis inhibitors/ICIs/chemotherapy/radiotherapy). All patients underwent intrathecal methotrexate via lumbar puncture or Ommaya reservoir. Thirty-two patients (91.43%) displayed malignant cells in CSF. The diagnosis of LM was established by CSF cytology alone in 10 patients (28.57%) and by both MRI and CSF cytology in 21 patients (60%). LM developed in 33 patients (94.29%) during the course of lung cancer, and LUAD was detected in 2 patients (5.71%) at the time of LM diagnosis. CSF intracranial pressure was decreased (≤ 200 mmH2O) in 57.14% of patients with LM. CSF abnormal biochemical measures also occurred in some LM patients.We retrospectively profiled 35 LUAD patients with LM for analysis. All patients had matched CSF and plasma samples. The demographic and clinical characteristics of the included patients are shown in Tables ). No somatic mutation was detected in the CSF and plasma of one patient. A total of 262 somatic variants of 103 mutated genes were detected in 35 CSF samples. Altogether, 85 somatic variants of 41 mutated genes were identified in 35 plasma samples, presenting a lower count than in CSF. For the detection of genomic alterations, only 37 (34.6%) were present in both plasma and CSF. However, 66 (61.7%) could be detected in CSF but could not be found in plasma, while 4 (3.7%) could be found in plasma but could not be detected in CSF , TP53  and CDKN2A  mutations frequently occurred in CSF, and coexisting TP53 and EGFR were found in 22  CSF samples. In addition, the most frequently altered genes in plasma were EGFR  and TP53 . Based on statistical analysis, the mutation frequencies of EGFR, TP53 and CDKN2A were significantly higher in CSF than in plasma  alterations in CSF, followed by CDKN2A  and MYC  mutations. However, only one CNV alteration was detected in the plasma sample. The percentage of CSF variants with CNV alterations was higher than that of plasma . Therefore, these results suggested that CSF had higher CNV alterations, and the importance of CNV in these alterations needs to be explored.To delineate the genetic landscape, the somatic mutations from the matched CSF and plasma ctDNA of 35 patients were analysed by applying an NGS panel Fig. . No somaTo further evaluate the genetic relevance and divergence between CSF and plasma, we analysed the shared and independently evolved mutations in each individual. In the 35 paired CSF-plasma pairs, a median 14.29% shared mutation rate was seen, which was relatively low. There were a few shared mutations found in 65.71% (23/35) of paired samples , a total of 16 patients did not receive osimertinib before the diagnosis of LM with baseline CSF and plasma genotyping. In cohort 2 (resistance), a total of 16 patients progressed after osimertinib and developed LM with CSF and plasma genotyping (Table EGFR tyrosine kinase inhibitor (EGFR-TKI). In cohort 2, gefitinib was the most common EGFR-TKI before the application of osimertinib. In cohort 2, 1 patient received osimertinib as first-line therapy, 13 patients as second-line therapy, and 2 patients as third-line treatment. In cohort 1, 87.5% (14/16) of the patients had EGFR exon 19 deletion or 21 L858R mutation in CSF, whereas only 50% (8/16) of the patients had these mutations in the matching plasma. In cohort 2, 81.25% (13/16) of the patients had EGFR exon 19 deletion or 21 L858R mutation in CSF, whereas only 50% (8/16) of the patients had these mutations in the matching plasma.We categorized patients with ng Table . In coho. EGFR T790M is the most common mechanism of resistance following treatment with 1st- or 2nd-generation EGFR-TKIs. However, in cohort 1 , NFE2L2, PKHD1 and POLD1 mutations were associated with shortened median OS  indicated a lower median OS. This result suggested that EGFR-mutated LUAD and CSF TERT mutation remained independent survival factors after adjusting for other patient characteristics.To assess the potential biomedical importance of genetic mutations, we examined their correlations with clinical variables. In further multivariate Cox proportional hazards regression analysis, patients with . The genetic profiles of CSF ctDNA demonstrated EGFR-independent resistance mutations  and Eastern Cooperative Oncology Group performance score (ECOG PS) progressed dramatically, and CSF ctDNA showed a higher frequency of ons Fig. B. UnfortEGFR and found that it is higher in CSF than in plasma. LM is more common in patients with EGFR mutations than in those with wild-type EGFR [EGFR-mutated patients, the detection rate of EGFR was 67.6–100% in CSF and 36.4–73.1% in plasma [EGFR was the most frequently mutated gene in CSF, accounting for 82.86%, which was higher than that in matched plasma. This is also consistent with Li’s findings [EGFR mutations may be more susceptible to BMs. In short, the available data revealed heterogeneous genetic profiles between CSF and plasma, with good concordance in driver mutations.Liquid biopsy is an alternative method to detect mutations in patients if tumour samples are not available. For LM patients, CSF and plasma can be easily obtained. In this research, we characterized the genomic alteration of ctDNA in the CSF and compared it to matched plasma samples. In our study, we revealed that CSF genetic profiles had a unique role in identifying patients with LUAD with LM and that plasma genetic profiles failed to do so. At the time of initial diagnosis of LM, more abundant genotypes were detected in CSF than in plasma in addition to driver mutations. Moreover, we demonstrated that CSF ctDNA has a significantly higher detection rate than plasma. This result is consistent with a previous study that showed that CSF ctDNA exhibits a more comprehensive genetic landscape of CNS metastases than plasma , 17, 20.ype EGFR . Moreoven plasma . In our findings , suggestIn TRACERx research, subclonal whole genome doubling (WGD) was detected in 29% of lung tumours. These data demonstrate that WGD and copy number heterogeneity were associated with shorter disease-free survival and distant metastases, respectively. WGD and copy number instability are important factors of relapse in NSCLC patients, which guide the evolution of clinical cancer . NumerouMYC, MMP13 or YAP, which are enriched for focal amplification in BMs, can each contribute to brain metastasis formation. TP53, CDKN2A, and TERT are abundant in a variety of metastatic cancers. While TP53 mutations are strongly associated with genetic instability, CDK2NA and TERT play a key role in regulating cell proliferation, and both pathways are frequently perturbed in metastatic tumours. In conclusion, these mutated genes may disturb pancancer hallmarks of tumorigenesis, hence improving the aggressiveness of the tumour [EGFR, TP53, MYC, CDKN2A and CDKN2B genes in CSF were significantly higher than those in LUAD tissue. Therefore, genomic characterization of the CSF of patients with LM represents a feasible strategy to find a potential method for the detection of metastasis.Studies have demonstrated the genetic heterogeneity between the original tumour and the metastatic lesions in the same patient . Genetice tumour , 27. To This conclusion was reached in the study of Nanjo et al. that the T790M mutation was less frequent in leptomeningeal than in extracranial specimens by biopsy of patients with lung cancer tissue and leptomeningeal metastases . This stEGFR C797S mutation, MET dysregulation, and TP53 plus RB1 co-occurrence were possible resistance mechanisms of LM in the progression of osimertinib of CSF in NSCLC [EGFR CNV. Furthermore, another study also revealed that EGFR amplification is the resistance mechanism associated with EGFR-TKIs [EGFR mutation was one of the most common acquired resistance mechanisms detected, followed by MET amplification [EGFR CNV occurred in 7 patients and MET mutations in 3 patients after the diagnosis of LM, which partly accounted for the progressive disease of LM. In our study, we found cell cycle pathway alterations  after osimertinib administration. In previous studies, altered cell cycle genes were found to be possibly involved in the mechanism of resistance to osimertinib as the 1st- or 2nd-line therapy [PIK3CA amplification or mutations promote tumour infiltration and activate the PI3K/AKT/mTOR pathway, which suggests that PI3K/AKT/mTOR pathway activation might be related to resistance to 3rd-generation EGFR-TKIs [PIK3CA mutation was present in the osimertinib-resistant cohort but not in the osimertinib-naive cohort.In the study by Zheng et al., in NSCLC . UnfortuGFR-TKIs . From thfication . In coho therapy . PIK3CA GFR-TKIs , 33. SimTissue biopsy of the primary tumour to determine the presence of SCLC transformation is also one of the resistance mechanisms to osimertinib . HoweverEGFR-mutated LUAD was an independent favourable predictor of survival, whereas TERT mutation in CSF was an independent predictor of poor survival after excluding other confounding factors. Patients with advanced LUAD who harboured EGFR mutations had significantly longer OS than those without EGFR mutations after treatment with EGFR-TKIs [EGFR mutations may be related to the use of EGFR-TKIs [EGFR-mutated LUAD were treated with EGFR-TKIs before the diagnosis of LM. The TERT gene represents a ribonucleoprotease that is essential for the replication of chromosome termini and telomere elongation in eukaryotes. The study suggests that targeting TERT promoter (pTERT) mutations may serve as a viable approach for cancer therapy [TERT mutations are associated with a poor prognosis in tumours, such as thyroid malignancies, melanoma and gliomas [TERT mutations were detected in 11% of patients with NSCLC, and TERT mutation carrier status was an independent risk factor for poor prognosis [TERT mutations were found in the CSF of 11.43% of patients with LM with LUAD, and these patients had a worse prognosis in our study. This had not been reported to be associated with survival in LM. Thus, we believe that TERT mutation may have clinical value as a potential biomarker for disease monitoring [In our study, a multivariate analysis indicated that the presence of GFR-TKIs . Suda etGFR-TKIs . This haGFR-TKIs . There i therapy , 39. Pre gliomas –43. Yangrognosis . Likewisnitoring , 46.TP53 mutation and EGFR/TP53 comutation have been considered poor prognostic factors in LUAD patients [TP53 mutations in CSF showed shorter OS than those in the other groups (P < 0.05). Dual EGFR/TP53 mutation was associated with inferior OS compared with dual EGFR/TP53 wild-type, although these results were not statistically significant. In this study, CDKN2A was common in CSF samples, accounting for 28.6%, regardless of prognosis. This is consistent with the findings of Yang et al. [In the previous literature, patients . In our g et al. .In the CSF circulation, disseminated cancer cells can float freely within the CSF or attach to the meninges and can be captured by CSF cytological examination or appear as linear or nodular enhancement on MRI . Remsik Finally, we demonstrated with a specific case that dynamic changes in CSF ctDNA at different stages could better predict intracranial tumour responses and track clonal evolution in LM patients.There are several limitations in our study. First, this was a retrospective study with a small sample size. Second, matched primary lung cancer tissues of the patients were unavailable, and the NGS data were obtained from CSF- or plasma-derived ctDNA without the analysis of matched tumour tissue DNA. Third, there is a lack of observation of CSF tumour markers, and we will continue this study in future observations. However, this is still a rare study that involved exploration of exactly matched CSF and plasma genetic information and analysis of survival prognosis in LUAD patients with LM.EGFR-mutated LUAD was associated with better OS, and CSF TERT mutation was associated with poorer OS. These indicators may have clinical value as potential novel biomarkers for disease monitoring.In conclusion, our findings demonstrate that CSF is a more sensitive and reliable liquid biopsy medium than plasma for LM. CSF ctDNA could provide a more comprehensive genetic landscape of LM, which reveals the potential metastasis-related mechanisms of malignant tumours and the resistance mechanisms to EGFR-TKIs and guides clinical strategies. In addition, Supplementary file1 (TIF 16644 KB)Supplementary file2 (TIF 21739 KB)Supplementary file3 (TIF 14230 KB)Supplementary file4 (DOCX 14 KB)Supplementary file5 (DOCX 14 KB)Supplementary file6 (DOCX 14 KB)Supplementary file7 (DOCX 13 KB)Below is the link to the electronic supplementary material.