Diffuse gliomas, and glioblastomas, in particular, represent a diagnostic and clinical challenge. Standard neuroimaging continues to have many limitations for accurate diagnostic assessment, resection planning and treatment follow-up. The present two-review series comprehensively summarizes recent evidence on hemodynamic imaging applications in the context of diffuse cerebral glioma. Part A provides an overview of the concepts underlying hemodynamic imaging modalities and critically discusses the diffuse glioma differential diagnosis and tumor grading results reported in the literature.Diffuse gliomas are the most common primary malignant intracranial neoplasms. Aside from the challenges pertaining to their treatment—glioblastomas, in particular, have a dismal prognosis and are currently incurable—their pre-operative assessment using standard neuroimaging has several drawbacks, including broad differentials diagnosis, imprecise characterization of tumor subtype and definition of its infiltration in the surrounding brain parenchyma for accurate resection planning. As the pathophysiological alterations of tumor tissue are tightly linked to an aberrant vascularization, advanced hemodynamic imaging, in addition to other innovative approaches, has attracted considerable interest as a means to improve diffuse glioma characterization. In the present part A of our two-review series, the fundamental concepts, techniques and parameters of hemodynamic imaging are discussed in conjunction with their potential role in the differential diagnosis and grading of diffuse gliomas. In particular, recent evidence on dynamic susceptibility contrast, dynamic contrast-enhanced and arterial spin labeling magnetic resonance imaging are reviewed together with perfusion-computed tomography. While these techniques have provided encouraging results in terms of their sensitivity and specificity, the limitations deriving from a lack of standardized acquisition and processing have prevented their widespread clinical adoption, with current efforts aimed at overcoming the existing barriers. Cerebral diffuse gliomas are the most malignant primary and frequently diagnosed intracranial tumors ,2. The mThese techniques and others have all been exploited to foster progress in pre-operative diffuse glioma assessment and follow-up, with the use of various multimodality approaches becoming the new standard of care ,9,19. HeIn normal physiology, cerebral vessel regulation and brain hemodynamics are highly sophisticated and fine-tuned by changes in blood gases, neuronal and tissue metabolism and blood pressure , and it Perfusion imaging follows the blood in the vascular system up to the tissue of interest using indicators (or tracers). These can be divided into two classes: intravascular, which remain in the vessels in physiological conditions, and freely diffusible, which can leave the intravascular space and diffuse throughout the entire tissue volume ,38. The DSC-MRI, DCE-MRI and PCT are the available CE perfusion imaging techniques ,56, and In this respect, it is important to note that in the literature, the term “relative”  is usually used to define semiquantitative parameters but the term “normalized”  can be used interchangeably. Confusion can arise between the terms “relative” and “regional”, with the second referring to absolute measurements. In the present work, unless otherwise specified, we refer to the relative semiquantitative parameters and the r/n prefix has been omitted to enhance readability.Two mechanisms govern the cerebrovascular system by controlling the vascular smooth muscle cell tones to help regulate the regional CBF: autoregulation, which ensures the maintenance of blood flow in response to changes in perfusion pressure , and metWith the introduction of end-tidal targeting gas delivery systems, the possibility granted by precise independent control of the end-tidal pressure of carbon dioxide and oxygen  has incrAfter having reviewed the basic principles of hemodynamic imaging, we now present how their application has been investigated in the clinical work-up of patients with high-grade gliomas and discuss the relevant differences in the assessed parameters among the different tumor entities.The first magnetic resonance presentation of a brain lesion showing a heterogeneously enhancing mass surrounded by extensive peritumoral edema on a T2-weighted image is not univocally interpretable. Hemodynamic imaging has been investigated with the aim of differentiating between gliomas and other possible neoplastic entities such as brain metastases (BM) ,99,100 aIn an ideal case scenario, metastases should always be distinguished pre-operatively from high-grade gliomas to ensure a complete patient diagnostic assessment and to optimize the treatment decision-making . BM, difThe distinction between gliomas and cerebral lymphomas is even more sensible, as the latter should be treated with chemoradiotherapy with the role of resection remaining controversial ,123. DSCFurthermore, cerebral abscesses can also sometimes be difficult to distinguish from high-grade gliomas as these also present as a CE rim surrounding a central necrotic core, with diffusion-weighted imaging being particularly useful for this purpose ,133. PerThe standard imaging of gliomas can lead to an educated guess on the expected tumor grade, but as some low-grade gliomas can present with contrast-enhancement and a number of atypical high-grade gliomas do not readily exhibit BBB disruption, hemodynamic imaging can further support the differentiation of these two entities. Low-grade gliomas are mostly characterized by native vessel co-option while the hypoxic angiogenic microenvironment of high-grade gliomas presents with an increased number of leaky vessels . These fTo overcome the limitations intrinsic to the various imaging modalities , efforts were made to achieve protocol standardization aimed at solving the variability of the reported methodologies and their resulting reported cut-offs ,218,219.Hemodynamic imaging-derived measurements are well suited to characterizing abnormal vasculature perfusion, BBB defects and providing complementary information with respect to standard neuroimaging to assist in diffuse cerebral glioma patient diagnostic workup. Several studies have assessed the potential of hemodynamic imaging for brain tumor differential diagnosis and tumor grading. They reported high sensitivity and specificity, especially of DSC but also of DCE and ASL-MRI for the differentiation of high-grade gliomas from brain metastases, cerebral lymphomas and non-neoplastic lesions, as well as for differentiating high-grade gliomas from low-grade gliomas. As recent efforts aim to tackle the standardization issues intrinsic to the imaging modalities investigated , a number of limitations have prevented the univocal interpretation of the previous findings, largely limiting a robust and widespread clinical translation.


Japan has faced numerous issues in the last twenty years with its mandatory long-term care (LTC) insurance program. This LTC insurance program obviously affected older adults’ informal support exchanges, reducing support from family and the community, which became more valuable, subjectively. Furthermore, changes in support have impacted older adults’ subjective well-being and children’s perceived care motivation. Additionally, a mandatory uniform system challenges the issue of tolerance of diversity, meaning how non-traditional families’ opinions be involved LTC situations. This symposium discusses unexpected shifting issues in Japan in the implementation of a public LTC program with a focus on older adults’ support exchanges. The first paper examines the long-term impacts of formal and informal support by examining the effects of implementing formal services. The second paper assesses a community’s role in relation to family in the presence of a public LTC program. The third paper examines the subjective impacts of older parents, who provided support to adult children and their reciprocal expectations of receiving LTC. The fourth paper, on the other hand, articulates reciprocal impacts on sons’ care motivation, which has become more important, since the introduction of the LTC program reinforced men’s participation in LTC. Finally, the fifth paper clarifies how a public uniform program accommodates informal support from non-traditional families when the program premises the presence of family in advanced care planning. Our findings have long-term implications for aging societies in relation to formal and informal support exchanges.


Classic small molecule inhibitors that directly target pathogenic proteins typically rely on the accessible binding sites to achieve prolonged occupancy and influence protein functions. The emerging targeted protein degradation (TPD) strategies exemplified by PROteolysis TArgeting Chimeras (PROTACs) are revolutionizing conventional drug discovery modality to target proteins of interest (POIs) that were categorized as “undruggable” before, however, these strategies are limited within intracellular POIs. The novel new degrader technologies such as LYsosome-TArgeting Chimaeras (LYTACs) and Antibody-based PROTACs (AbTACs) have been successfully developed to expand the scope of TPD to extracellular and membrane proteins, fulfilling huge unmet medical needs. Here, we systematically review the currently viable protein degradation strategies, emphasize that LYTACs and AbTACs turn a new avenue for the development of TPD, and highlight the potential challenges and directions in this vibrant field. Remarkable advances in human genetic studies have revealed a broad spectrum of protein targets that associate with disease progression Targeted protein degradation (TPD) has emerged to be a powerful tool to handle these undruggable targets and exhibits significant therapeutic benefit over standard small-molecule inhibition strategy To broaden the spectrum of target proteins, researchers are turning their attentions on the lysosome degradation system. Two marvelous approaches, termed LYTACs Over the last two decades, the breakthrough progress in TPD technologies not only provides useful tools for biological discovery, but also offers viable therapeutic candidates in clinic. We herein summarize the novel technologies with different mechanisms, as shown in Figure  in vivoA PROTAC molecule consists of a targeting warhead for intracellular POI and an E3 ligase ligand for recruitment of E3, which are connected by a flexible linker In addition to the extensively utilized UPS in current TPD approaches, novel techniques such as AUTAC and ATTEC have been developed to modulate and control protein levels by harnessing the autophagy/lysosome pathway  Similar to AUTAC based on autophagy-lysosome system, ATTEC is a linker compound that tethers the POI to the autophagosome by interacting with both POI and LC3 proteins Despite the promising prospect of TPD strategy, non-cytosolic proteins lied beyond the scope of TPD for a long time, which limited their further application. Encouragingly, the novel technologies, LYTAC and AbTAC, have emerged to broaden the spectrum of protein targets.LYTAC is a novel technology that targets extracellular and/or membrane protein to induce degradation by harnessing the endosome/lysosome pathway. It is a bifunctional conjugate that simultaneously binds the extracellular domain of a target and a cell-surface lysosome-targeting receptor (LTR) to form a ternary complex, leading to protein internalization via clathrin-mediated endocytosis Bispecific antibodies (bsAbs) refer to a large family of molecules that recognize two different epitopes or antigens To fully understand the discussed techniques and choose the appropriate one for the problem at hand, we compare the advantages and disadvantages of intracellular protein degradation strategies and the two extracellular protein degradation approaches (LYTAC and AbTAC), as shown in Table To date, LYTAC and AbTAC have been the main two TPD technologies for extracellular and membrane protein degradation. Although they are recognized as tremendous milestones in the development of TPD, targeting extracellular protein degradation is still in its infancy. In this part, we mainly summarize the design principles, recent progress, and current synthesis strategies of LYTAC and AbTAC, providing theoretical basis for the future application. By focusing on the successive conceptual and technical innovations in this field, we hope to encourage and inspire further efforts to optimize current techniques and create new TPD methods.LYTAC is designed with one end binding to the POI and the other end recognized by LTR. The incorporation of a ligand for efficient drug delivery via a LTR has been widely explored Cation-independent mannose-6-phosphate receptor (CI-M6PR), one member of cell-surface LTR families, can enclose hydrolases or other cargoes in endosomes, and subsequently deliver those proteins into lysosomes for depletion M6Pn glycopolypeptides synthesis strategy starts with the conversion of mannose pentaacetate to M6Pn-NCA in 13 steps and ends with copolymerization of M6Pn-NCA to acquire poly(M6Pn) polypeptides Unlike CI-M6PR, Asialoglycoprotein receptor (ASGPR) is a LTR which is highly expressed in hepatocytes The degradation ability of LYTAC also relies on the affinity between POI and its ligand. For targeting the significant disease-associative proteins, there are three choices for POI ligands, including antibodies, small molecules, and peptides Chemically, a LYTAC molecule can be assembled via azide-alkyne cycloaddition (click chemistry) between a TLR ligand and a POI ligand. For example, following modular design and synthesis, a POI ligand labeling with azide group is conjugated with a TLR ligand with alkynylation via copper-free strain-promoted click reaction, in turn, it also works that a POI ligand labeled with DBCO (dibenzocyclooctyne) or BCN can conjugate to an azide-labeled TLR ligand. After the conjugation reaction, the product is interrogated by native gel electrophoresis As previously described, the AbTAC acts by assembling separately expressed half IgGs to form a bispecific IgG for degrading cell-surface proteins via RNF43 The production of antibodies binding to RNF43 and POI begins with the phage display technique CI-M6PR is highly expressed in fetal and neonatal tissues but it decreases postnatally; however, it is overexpressed in some pathological conditions, such as fibrosis and some cancerous diseases AbTAC represents a new archetype to LYTAC with the ability to deplete cell-surface proteins. Despite its great promise in protein degradation, it is unclear how AbTAC recruit membrane-bound E3 ligases to induce cell-surface POI degradation via lysosome pathway. Only when the detailed regulation process is elucidated, could we make good use of this degradation pathway better. Moreover, a potential problem is whether there is appropriate distance between half IgGs for binding of membrane-bound E3 ligase and POIs at the same time. In this case, we envision that a bifunctional peptide with two warhead and a flexible linker can be used to target transmembrane E3 ligase and POIs simultaneously. We can also utilize phage display to rapidly generate targeting peptide for membrane-bound E3 ligases with high affinity and high specificity Currently, the number of E3 ubiquitin ligase ligands used in PROTAC technology is limited, which restricts their subsequent application and development. Also, it was verified that the loss of core components of E3 ligases leads to resistance to PROTACs LYTAC provides possibilities to change protein ligands into degraders. TPD strategy possesses kinetic advantages by binding to any “nook” or “cranny” on POIs with low dose Drug resistance and relapse are two major obstacles in cancer treatment Antibody-drug conjugates (ADC) are monoclonal antibodies conjugated to cytotoxic agents by environment-responsive linkers, which enable traditional drugs to have high tumor specificity and potency Because LYTACs are macromolecules different from those of traditional protein-targeting small molecules, their aqueous solubility, drug delivery, and pharmacokinetics, and adverse reaction remain elusive. We estimate that the universal expression of cell-surface LTR and the instability of LYTACs may cause off-target effect and short drug half-life. Nanoparticles (NPs) have emerged as a powerful drug-delivery tool to enhance the specificity of drug actions, while reducing the systemic side effects Nowadays, TPD has been recognized as a game-changing strategy by inducing the degradation of the 'undruggable' targets and PROTACs have broken the classical Lipinski's rule of five In conclusion, by focusing on TPD strategies towards extracellular and membrane proteins, we hope to provide readers with a resource to help navigate in this booming field, and inspire further efforts to create new degradation modalities.


This proof-of-concept study demonstrates that blood-based liquid biopsy using next generation sequencing of cell-free DNA can non-invasively detect multiple classes of genomic alterations in dogs with cancer, including alterations that originate from spatially separated tumor sites. Eleven dogs with a variety of confirmed cancer diagnoses  who were scheduled for surgical resection, and five presumably cancer-free dogs, were enrolled. Blood was collected from each subject, and multiple spatially separated tumor tissue samples were collected during surgery from 9 of the cancer subjects. All samples were analyzed using an advanced prototype of a novel liquid biopsy test designed to non-invasively interrogate multiple classes of genomic alterations for the detection, characterization, and management of cancer in dogs. In five of the nine cancer patients with matched tumor and plasma samples, pre-surgical liquid biopsy testing identified genomic alterations, including single nucleotide variants and copy number variants, that matched alterations independently detected in corresponding tumor tissue samples. Importantly, the pre-surgical liquid biopsy test detected alterations observed in spatially separated tissue samples from the same subject, demonstrating the potential of blood-based testing for comprehensive genomic profiling of heterogeneous tumors. Among the three patients with post-surgical blood samples, genomic alterations remained detectable in one patient with incomplete tumor resection, suggesting utility for non-invasive detection of minimal residual disease following curative-intent treatment. Liquid biopsy allows for non-invasive profiling of cancer-associated genomic alterations with a simple blood draw and has potential to overcome the limitations of tissue-based testing posed by tissue-level genomic heterogeneity. Non-invasive blood-based genomic profiling, often called liquid biopsy, is increasingly employed in human medicine to support cancer diagnosis and management , 2. It rKIT gene) are frequently found in mast cell tumors  specifically developed for the non-invasive detection, characterization, and management of cancer in dogs. This is the first report of a next-generation sequencing (NGS) based multi-cancer early detection test designed to simultaneously interrogate multiple classes of cancer-associated genomic alterations, including single nucleotide variants (SNVs), insertions and deletions (indels), translocations, and copy number variants (CNVs), in cfDNA extracted from canine blood samples. This proof-of-concept study aims to demonstrate the potential of liquid biopsy to broadly profile tumor-derived genomic alterations, and to capture the genomic heterogeneity of multiple, spatially separated tumor clones (within the same primary site or across different metastatic sites) that would be missed by traditional single-site tissue biopsy, suggesting broad utility as a diagnostic tool in veterinary oncology.Eleven client-owned dogs with a variety of confirmed cancer diagnoses  that were scheduled for surgical resection, and five dogs presumed to be cancer-free , were enrolled across multiple care settings. All cancer subjects were enrolled under a dedicated protocol that was independently approved by the institutional animal care and use committee (IACUC) or internal review board at two veterinary centers, and all cancer-free subjects were enrolled under a separate dedicated protocol that was independently approved by the institutional clinical review board at one veterinary center; the protocols were also approved by institutional clinical review boards at the other participating sites, based on each site's requirements. All subjects were client-owned, and written informed consent was obtained from all owners.From each patient, a baseline blood sample was collected at the time of enrollment. In 9 of the 11 patients with cancer, samples of resected tumor specimens from one or more sites within each primary tumor, and from one or more metastatic sites if available, were collected during the subsequent surgery. In three cancer patients, a post-surgical blood sample was collected 10, 15, and 189 days after surgery, respectively. Clinical and demographic data, plasma volumes, and cfDNA yields for all enrolled subjects are summarized in Blood samples were processed with a double-centrifugation protocol to separate plasma from white blood cells (WBCs) –23. CfDNAll sequencing reads were aligned to the CanFam3.1 reference genome , and somIn the nine cancer patients that had tumor tissues analyzed, all nine had somatic alterations detected in tissue, based on aggregate variant calls across all available tissue samples . In 5 ofTP53, KRAS, EGFR, and PIK3CA that are frequently associated with cancer in both humans and dogs   , 29, whiGenomic profiling of spatially separated tumor tissue samples from the same subject demonstrated genomic heterogeneity across sites within the primary mass as well as across distant sites, in keeping with the polyclonal nature of cancer ; and theNRAS, one SNV in KRAS, and two distinct SNVs in TP53, were identified across four of the five tissue samples  detected, suggesting that profiling multiple classes of genomic alteration improves overall detection of tumor-derived cfDNA compared to relying on just one class. Among patients for whom cancer was not detected in blood, all had localized disease; these results are consistent with the observation in humans that the proportion of patients with detectable tumor-derived cfDNA in plasma is higher in metastatic disease compared to localized disease . Larger Furthermore, our results suggest utility for liquid biopsy in detecting minimal residual disease (MRD) following curative-intent surgery. After a complete resection, tumor-specific genomic alterations should no longer be present in plasma cfDNA, whereas such alterations would persist—potentially at lower levels—following an incomplete resection.The ability of liquid biopsy to detect genomic alterations originating from one or multiple tumor sites within a patient offers several advantages compared to traditional tissue-based testing. First, it is non-invasive and thus poses a lower risk to patients compared to biopsy or surgery. Second, it allows for a more comprehensive characterization of the full genomic landscape of the patient's malignancy, across multiple sites within the primary tumor and across metastatic deposits. This tissue-level heterogeneity, which reflects the polyclonal nature of most clinical cancers, would be otherwise difficult to evaluate in dogs due to the morbidity and expense associated with obtaining multiple tissue samples from the primary as well as metastatic sites. Furthermore, obtaining multiple tissue samples is generally not performed in dogs with gross metastatic disease, as aggressive therapy is not typically pursued in these individuals, and the invasiveness of collecting samples from metastatic lesions would cause an unacceptable level of morbidity in a patient with advanced cancer . A bloodLiquid biopsy testing can support multiple applications across the continuum of canine cancer care, including: screening (for early detection) in high-risk populations, aid in diagnosis, detection of MRD, selection of targeted therapies, and monitoring for cancer recurrence or for treatment response by serial testing; and promises to bring the power of precision oncology to veterinary practice through a simple blood draw that does not require changes to the clinical routine . FinallyWidespread access to multi-cancer early detection testing would be highly beneficial for canine patients, given that cancer is by far the leading cause of death in dogs . The curBlood-based liquid biopsy testing—first used for non-invasive prenatal testing  and morescience@petdx.com.Restrictions apply to the datasets: The datasets presented in this article are not readily available because they contain proprietary information. Requests to access the datasets should be directed to All cancer-diagnosed subjects were enrolled under a dedicated protocol, which was independently reviewed and approved by the institutional animal care and use committees (IACUCs) at the University of Guelph Ontario Veterinary College and the University of Minnesota Veterinary Medical Center, as well as by institutional clinical review boards at additional sites based on each study site's requirements; all cancer-free subjects were enrolled under a separate dedicated protocol, which was independently approved by the Veterinary Centers for America (VCA) Clinical Studies Institutional Review Board, as well as by institutional clinical review boards at additional sites based on each study site's requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.KK, JC, DG, and AF contributed to the conception and design of the study. LM, RM-P, LH, and AF managed the logistics of patient recruitment and sample collections. KK, PN, GH, DF, JT, IC, and DT generated the data and performed data analysis. KK and DT wrote the first draft of the manuscript. All authors contributed to manuscript revisions and approved the submitted version.All authors are employed by or affiliated with PetDx; and receive compensation from PetDx and/or hold vested or unvested equity in PetDx.


ABSTRACT IMPACT: The findings from this study will inform the development of an FDA-approved patient-rated outcome measure of drug and alcohol craving that can be used in clinical trials aimed at developing or testing effective treatments for substance use disorder. OBJECTIVES/GOALS: Craving is a potential target of investigative medications to reduce drug use due to the strong link between craving and drug use. We will identify all existing craving measures as the first step for developing an FDA-approved patient-rated outcome measure for use in clinical trials. METHODS/STUDY POPULATION: Following PRISMA guidelines, we will update Rosenberg’s (2009) craving review by conducting a systematic review of all existing published and unpublished measures of craving for alcohol, nicotine, cannabis, opioid, and stimulant use. Electronic database , forward, backward, and author searches will be conducted. We will also request unpublished craving measures on major listservs . All papers included in Rosenberg’s (2009) review through September 2020 will be included. RESULTS/ANTICIPATED RESULTS: The findings from this review will provide a comprehensive summary of the construct of craving and its hypothesized and tested domains. This review will elucidate whether the literature suggests there are components of craving unique to alcohol, nicotine, cannabis, opioid, and/or stimulant use, and whether there are key elements of craving common across the disorders. Therefore, these findings will inform whether a single patient-rated outcome measure of craving can be developed for use across substances or if unique patient-rated outcome measures of craving need to be developed for each substance. DISCUSSION/SIGNIFICANCE OF FINDINGS: While many different measures of craving exist, none have gone through the developmental steps required to qualify as an FDA-approved patient-rated outcome measure on which drug treatment labeling can be based. Completing this systematic review is the first step in this process.


In the past 20 years, Cardiac Computed Tomography (CCT) has become a pivotal technique for the noninvasive diagnostic work-up of coronary and cardiac diseases. Continuous technical and methodological improvements, combined with fast growing scientific evidence, have progressively expanded the clinical role of CCT. Recent large multicenter randomized clinical trials documented the high prognostic value of CCT and its capability to increase the cost-effectiveness of the management of patients with suspected CAD. In the meantime, CCT, initially perceived as a simple non-invasive technique for studying coronary anatomy, has transformed into a multiparametric “one-stop-shop” approach able to investigate the heart in a comprehensive way, including functional, structural and pathophysiological biomarkers. In this complex and revolutionary scenario, it is urgently needed to provide an updated guide for the appropriate use of CCT in different clinical settings. This manuscript, endorsed by the Italian Society of Medical and Interventional Radiology (SIRM) and by the Italian Society of Cardiology (SIC), represents the first of two consensus documents collecting the expert opinion of Radiologists and Cardiologists about current appropriate use of CCT. Cardiac Computed Tomography (CCT) was historically adopted as a tool to rule-out coronary artery disease (CAD) due to the well-established very high negative predictive value. Recently, the results of multicenter randomized clinical trials have changed the perception of CCT in the clinical world, leading the scientific community to recognize CCT as the first line diagnostic test for most of the patients with suspected chronic coronary syndrome  and in sIn this complex and revolutionary scenario, in which guidelines help the translation of evidences into clinical practice , there iThis manuscript, endorsed by the Italian Society of Medical and Interventional Radiology (SIRM) and by the Italian Society of Cardiology (SIC), represents the first of two consensus documents collecting the expert opinion of Radiologists and Cardiologists about current appropriate use of CCT and integrates the guidelines for appropriate use of cardiovascular magnetic resonance (CMR) recently published by the same working group .The writing committee discussed the table of content and assigned referrals for each chapter.A.Strong recommendation: there is evidence, general agreement, or both, that the test is useful (benefit >  >  > risk).B.Moderate recommendation: there is conflicting evidence or opinion about the usefulness of the test; the weight of evidence/opinion, however, is strongly in favor of the test’s usefulness. (benefit >  > risk).C.Weak recommendation: the test’s usefulness is less well established; there is a small net benefit (benefit ≥ risk)D.No recommendation: there is evidence or general agreement that the risk/harm outweighs benefits (benefit = or < risk).E.Expert opinion: there is insufficient evidence or evidence is unclear or conflicting, but this is what the working group recommends. Further research is recommended in this area.Each referral conducted literature review and drafted the assigned section highlighting indications and rating them according to the following score:Assigned scores were discussed in consensus by all authors and unanimously approved.In pre- and post-surgical complex congenital heart diseases (CHD), multimodality imaging is required for both the detailed evaluation of cardiovascular anatomy and for the functional characterization of cardiac chambers and flows. Catheterization is required for pulmonary vascular resistances calculation, whereas for most types of CHD and congenital coronary artery anomalies (CAAs) CCT is adopted as a complementary imaging modality  Table 11.Table 1Echocardiography is the initial imaging tool for morpho-functional evaluation; however, a frequently limited acoustic window hampers the assessment of the mediastinal vessels, extra-cardiac surgical conduits, and intra-cardiac complex anatomy, particularly in adults with grown-up congenital heart diseases (GUCH).The use of CCT has been described in patients of all ages and with CHD of all levels of complexity, especially when echocardiography is not exhaustive. CCT is generally recommended in complex conditions that require investigation of coronary vessels or complex vascular and thoracic anatomy , 16. CCTIn patients with suspected vascular rings and slings or tracheobronchial narrowing for complete cartilaginous rings, CCT is the method of choice for the pre-surgical evaluation of tracheobronchial tree and pulmonary parenchyma . CongeniCMR remains the method of choice in the follow-up of complex CHD due to the absence of ionizing radiation and for its capability to quantify vessel flows and ventricular function and to identify myocardial fibrosis. However, CMR is time consuming and image quality may be reduced in patients with metallic devices. CCT provides better visualization of stents, conduits, and metallic objects and is safe in patients with implanted pacemakers and defibrillators . MoreoveFinally, CCT may provide useful morphological information to avoid external coronary artery compression related to device release in transcatheter pulmonary valve replacement  and to iThe main limitation of CCT is ionizing radiation exposure; however, low-dose acquisition protocols can be adopted .Coronary Artery Calcium Score (CACS), reported as Agatston score , measureRecent studies , 29 haveCACS may have a role also in individuals aged 45-to-75 years with low cardiovascular risk but with strong family history of premature CAD and in diabetics patients aged > 40 years or at intermediate-risk of early CAD , 32.The absence of CACS carries a favorable 5-year and 15-year prognosis for patients with and without diabetes, respectively .Finally, CACS is considered useful for guiding preventive medical therapy , avoidinAccording to 2019 ESC guidelines on chronic coronary syndromes, coronary CT Angiography (CCTA) is not recommended for extensive screening of asymptomatic individuals . MoreoveThis is reflected in 2019 ESC guidelines that suggest that asymptomatic diabetic subjects with CACS > 400 may be referred for functional imaging or CCTA . HoweverFinally, some evidence suggests that CCTA could enhance screening in asymptomatic individuals in specific sporting  , 44 or wNon-cardiac surgery is associated with an incidence of complications from 7 to 11%, with a mortality rate of 0.8% to 1.5%, largely driven (42% of cases) by cardiac complications. Based on the rate of cardiovascular events , surgical procedures are classified at low, intermediate, or high risk . The current guidelines recommend coronary functional testing for patients with an unknown or impaired functional status undergoing intermediate-to-high risk non-cardiac planned surgery , 47. NevIn a recently published meta-analysis, CCTA was found to safely predict freedom from perioperative MACEs in a cohort of patients at high risk according to clinical indices . The sevCAD needs to be screened in patients scheduled for cardiac surgery for pre-operative risk assessment. In particular, a thorough cardiological evaluation is indicated in patients with severe valve disease with history of CAD, suspected ischemia, systolic dysfunction, male with age > 40 years, post-menopausal women, and patients with one or more risk factors. Several studies indicated that CCTA can reliably replace invasive coronary angiography (ICA) as a screening tool before valve interventions –52, espeIn the recent past, stable symptomatic patients with chest pain were non-invasively assessed using different functional tests, including mainly treadmill testing, stress echocardiography and single photon emission computed tomography (SPECT). Stress perfusion cardiac magnetic resonance (stress-CMR) and positron emission tomography (PET) were less used due to availability and costs concerns, albeit showing a higher diagnostic accuracy. Unfortunately, despite routine use of these tests, only one-third of the patients with a positive functional test turns out to be affected by obstructive CAD at ICA , revealiIn the recent years, CCTA was found to detect with high accuracy non-obstructive CAD defined by ICA, and to reduce unnecessary ICAs when compared to functional testing .CT), it is comparable to ICA with invasive FFR in targeted revascularization , be, be61], In past studies adopting old technology, it was found that the accuracy of CCTA was influenced by the pre-test probability (PTP) of CAD , being pHowever, CCTA is to be avoided in the presence of conditions which cannot ensure good image quality related to local availability and expertise, scanner technology, and patient characteristics, including extensive coronary calcification, irregular heart rate, severe obesity, and inability to breath-hold (Class III) .CCTA has the unique capability to non-invasively quantify coronary atherosclerosis and to characterize plaque morphology and composition with high accuracy compared to histology and intravascular ultrasound (IVUS) . This isPatients with obstructive CAD have worse outcomes compared to patients with nonobstructive or absent CAD . HoweverOther plaque characteristics, such as extent and location , have been associated with clinical outcome , 83, andThese data support the reporting of HRP features presence (if more than 2 HRP features are evident) even for non-obstructive lesions, as suggested by CAD-RADS guidelines .Some technical limitations may impact on CCTA-based plaque characterization, most importantly spatial resolution and other factors such as a certain degree of density overlap in lipid-rich and fibrous-rich non-calcified plaques. Dual-energy CT may overcome these limitations thanks to tissue decomposition algorithms. However, this approach is still limited to research and initial results need to be validated .Pericoronary adipose tissue is emerging as an imaging biomarker to identify plaque instability. Increased epicardial adipose tissue (EAT) attenuation was detected around inflamed plaques . MoreoveCT and stress-CTP allow to integrate information about the hemodynamic significance of coronary lesions to angiographic evaluation of CAD, thus potentially avoiding additional examinations and costs. In fact, data coming from iFFR show that only 35% of anatomically obstructive lesions have positive iFFR reflecting hemodynamic significance of a stenosis. Thus, iFFR is a key parameter to guide revascularization, improving the outcome and reducing health care costs [FFRre costs , 92  due to software constraints and offsite analysis [CT is strictly related to image quality. Imaging artifacts caused by low contrast, cardiac and respiratory motion, blooming due to severe calcification, and image noise due to low radiation exposure or high body mass index hamper FFRCT performance [Despite the advantages, FFRanalysis  and currformance .CT [Also, stress-CTP is capable of detecting functionally relevant stenosis, improving the diagnostic performances of CCTA, with similar performance in comparison with CCTA combined with FFRCT .Stress-CTP depicts perfusion defects as a hypo-attenuating myocardial region using either a static protocol (single scan acquired both at rest and during stress at the peak of iodine concentration in the coronaries) or a dynamic protocol in which several datasets are acquired during first pass perfusion. Dynamic stress-CTP has the advantage of providing quantitative evaluation of perfusion by estimating the myocardial blood flow .Regardless of the acquisition protocol, stress-CTP requires the administration of pharmaceutical stressors and notably increases both radiation exposure and iodinated contrast agent dose.Stress-CTP shows similar performance with respect to stress-CMR  and slightly better performance than single photon emission computed tomography (AUC 0.91 vs. 0.87)  and has CT, data on clinical utility and outcome have not been reported [However, these data need to be confirmed on larger populations. Furthermore, differently from FFRreported .


Low ovarian reserve is a serious condition, leading to sterility in up to 10% of women in their mid-thirties. According to current knowledge, serum anti-Müllerian hormone (AMH) levels for age are the best available marker for the screening the quantity of a woman’s functional ovarian reserve, better than age alone or other reproductive markers. This review summarizes recent findings, clinical utility and limitations in the application of serum AMH testing as an accurate marker for the screening of functional ovarian reserves and predicting age at menopause. AMH assessment hold promise in helping women make informed decisions about their future fertility and desired family size. However, screening of the functional ovarian reserve could be offered to all women at 26 years of age or older who seek to assess future fertility or in case of personal request, ovarian reserve screening may be considered beyond 30 years; however, it has never been advocated beyond 35 years, since it is not advisable to delay childbearing beyond this age. In this respect, an age-specific serum AMH levels lower than the 10th percentile may be used as a threshold for the identification of a low functional ovarian reserve in an individual woman. Its level should be interpreted with caution in the adolescent and young women aged below 25 years (since AMH levels peak at this age); recent users of hormonal contraceptives (since AMH levels transiently decrease until two months after discontinuation); and women with PCOS . However, the ability of AMH levels to predict the time to menopause is promising but requires further investigation and routine AMH testing for the purposes of predicting the time to menopause is not recommended. Societal shifts triggered by a greater focus on education and career opportunities, developments in assisted reproduction, rise of effective contraception, value changes, gender equity, partnership changes, lack of suitable partners, housing conditions, economic uncertainty, and the absence of supportive family policies have resulted in a trend toward delayed childbearing worldwide . It has Assessment of functional ovarian reserves hold promise in helping women make informed decisions about their future fertility and desired family size, rather than simply providing them with generic age-related fertility recommendations -6.Here, we discuss the clinical utility and limitations of anti-Müllerian hormone (AMH) testing as an accurate marker for the screening of functional ovarian reserves and predicting age at menopause.The functional ovarian reserve defines the quantity and quality of the ovarian primordial follicular pool, which declines with age in women, with a more rapid decline after the mid-30s until it reaches undetectable levels at menopause . AlthougLow ovarian reserve is a serious condition, leading to sterility in up to 10% of women in their mid-thirties, and is mostly irreversible . The conEarly diagnosis is critical to help women make informed decisions about their plans for natural conception, opt for oocyte vitrification, or use donated sperm if single, while these alternatives still have reasonable prospects of success . AlthougOver the years, numerous parameters have been proposed to estimate the functional ovarian reserve. Serum follicle-stimulating hormone (FSH) and antral follicle count (AFC) have been widely applied in reproductive medicine for years. Beyond their general predictive value, both have specific disadvantages. FSH levels fluctuate during the inter/intra cycle of menstruation; tend to increase after 35 years of age; are dependent on the functional hypothalamic-pituitary-ovarian (HPO) axis; and have low sensitivity when the number of follicles significantly decreases. AFC has various sensitivities owing to technical limitations such as interobserver variability and ultrasound machines, which are restricted to antral follicles of measurable size; has different methodologies for counting antral follicles and intercycle variation in obese women; and may not be feasible for virgin girls in some cultures, if using the vaginal approach. Compared to these other parameters, serum AMH is currently the best proxy for assessing the ‘functional ovarian reserve’, which supports normal follicle progression and ovulation -13.Anti-Müllerian hormone is specifically expressed by small growing follicles in the ovary and has an inhibitory effect on primordial follicular recruitment and FSH-induced preantral follicle growth  25]. Fr. Fr25]. Its utility is further extended as a marker of ovarian responsiveness in assisted reproduction, ovulatory disorders such as polycystic ovary syndrome (PCOS), before or after ovarian damage by surgery, chemotherapy, or radiotherapy, as well as prediction and diagnosis of early menopause and premature ovarian insufficiency. In addition, given the specific site of production of AMH, its measurement has been used as a very precise marker of tumours originating from granulosa cells in female and Sertoli cells of the testis in males . Moreove•In cases of diminished functional ovarian reserves, individual risk assessment should be performed based on repeat confirmatory AMH testing, day 3-5 FSH measurement, and ultrasound findings of AFC. Final diagnosis should be made after complete evaluation of all results, in the context of any known individual risk factors for low ovarian reserves . Women w•In women with borderline diminished functional ovarian reserves, annual follow-up ovarian reserve testing is recommended. In addition, the restriction of factors that can increase the rate of decline, including impacts of certain lifestyles such as alcohol abuse, smoking, malnutrition, obesity, and stress should be highlighted.•In women with acceptable AMH levels, it should be emphasised that advanced maternal age is related to low quality of oocytes and is therefore strongly associated with an increased risk of aneuploidy and other adverse feto-maternal or neonatal outcomes.According to current knowledge, age-specific serum AMH levels, which are measured by Automated and therefore more accurate AMH assays , are theIt is important to remember that individual conditions can affect AMH values, and these should be considered prior to their clinical application and interpretation. Serum AMH levels do not peak until approximately 25 years of age; hence, care must be taken when interpreting AMH levels in adolescents and young women. In addition, hormonal contraceptive usage, particularly in long-term users, could transiently decrease serum concentrations of AMH by 20-30% but tend to recover after two months of discontinuation. Therefore, AMH should be measured at least 2 months after the discontinuation of hormonal contraceptives. Moreover, due to an increase in the number of pre-antral and small antral follicles, which primarily produce AMH, the serum concentration of AMH tends to be higher in women with PCOS compared to non-PCOS counterparts. Although this needs to be precisely defined,  the threFinally, women must be empowered to make voluntary and informed choices about whether to get tested based on objective information about all aspects of this test. Healthcare professionals may tend to focus on the implications of poor results, but the information provided should also include full aspects of both poor or acceptable test results and subsequent steps of action prior to the test is ordered.Age-specific AMH levels may provide a fairly reasonable prediction of age at menopause , particularly for women who are younger than 40 years, even if they still have regular menstrual cycles and normal levels of other reproductive hormones , 22. It Mathematical modelling has already been used to predict age at menopause. For example, a 30-year-old woman with an AMH level close to 1.1 ng/mL (7.85 pmol/L) is known to have a predicted mean age at menopause of 45  years ; a serumHowever, based on available studies  as well There were different commercial enzyme-linked immunosorbent assay (ELISA) kits used to measure AMH, which is varied in antibody pairs, standard curve ranges, and limits of detection  Table 3Table 3. •The age-dependent decline rate of AMH levels varies among women. An international guideline regarding age-specific AMH diagnostic thresholds for screening functional ovarian reserves or predicting age at menopause remains to be addressed.•Some endogenous and exogenous factors could influence serum AMH levels, which limit the accurate interpretation of AMH values in a clinical setting.•The predictive value of AMH for successful clinical pregnancy  is less promising; and therefore, AMH levels should not be used as a fertility test.•AMH assessment using manual enzyme-linked immunosorbent assay (ELISA) has some limitations due to intra-assay/interassay differences and requires careful sample preparation and storage. However, automated AMH assay platforms have greater precision, faster turnaround time, greater sensitivity, and accessibility in many countries and should be used as a standard method for AMH measurement.AMH has the following limitations:In conclusion, considering the precise results, the ease of a serum-based test, independent of specific time in the cycle serum, independent of hypothalamic pituitary function and very low inter-observer variability, AMH is the preferred ovarian reserve marker. However, lack of international guideline regarding age-specific AMH diagnostic thresholds for screening functional ovarian reserves or predicting age at menopause could limit the proper interpretation of AMH values in a clinical setting and remain to be addressed in future researches.Ultimately, further research is needed to understand whether releasing/restricting various factors that could affect ovarian status and also the optimal approaches that promote ovarian reserve among women.


Oxidative stress is a well-known common pathological process involved in mediating acute neurological injuries, such as stroke, traumatic brain injury, epilepsy, and hypoglycemia-related neuronal injury. However, effective therapeutic measures aimed at scavenging free reactive oxygen species have shown little success in clinical trials. Recent studies have revealed that NADPH oxidase, a membrane-bound enzyme complex that catalyzes the production of a superoxide free radical, is one of the major sources of cellular reactive oxygen species in acute neurological disorders. Furthermore, several studies, including our previous ones, have shown that the inhibition of NADPH oxidase can reduce subsequent neuronal injury in neurological disease. Moreover, maintaining appropriate levels of NADPH oxidase has also been shown to be associated with proper neurogenesis after neuronal injury. This review aims to present a comprehensive overview of the role of NADPH oxidase in neuronal death and neurogenesis in multiple acute neurological disorders and to explore potential pharmacological strategies targeting the NADPH-related oxidative stress pathways. Acute brain injuries, such as strokes, traumatic brain injury (TBI), epileptic seizures, and hypoglycemia-induced damage, all result in a substantial disease burden for both individuals and society at large. Though these injuries may have different neurological manifestations, a common pathophysiological process is cellular oxidative stress, which is largely induced by the excessive production of reactive oxygen species (ROS). For the past few decades, many studies focused on the role of ROS-generating pathways in various neurological disorders and led to the identification of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases as one of the major sources of ROS. While NADPH oxidase (NOX) contributes to normal physiological processes in the central nervous system, excessive activation of NOX results in neuronal damage in the setting of acute brain injury. In this current review, we summarize the evidence supporting the pathophysiological roles of NOX after acute stroke, TBI, epileptic seizure, and hypoglycemia. In addition, we explore the importance of NOX balance regarding the neurogenesis process after such brain injuries.2) is transported to the intracellular space, and hydrogen (H+) is exported to the extracellular space. Hydrogen peroxide and ROS, including hydroxyl radicals, are also produced as a result of this reaction. The superoxide produced by NOX plays an important role in killing foreign substances, but excess superoxide can lead to oxidative stress and cell damage. Numerous studies have shown that the NOX system is responsible for the production of ROS [The NOX family consists of membrane-bound enzymes that transport an electron from NADPH to oxygen, thereby producing superoxide free radicals . In thisn of ROS ,4. The Nn of ROS ,6,7,8,9.n of ROS . The dis2), which must be supplied to the living organism, is an essential molecule for survival. However, the fact that oxygen induces oxidative stress in metabolic processes in vivo and that the ROS produced therein damages cells or tissues has already been confirmed through many studies. In vivo oxidative stress is a phenomenon that occurs when the balance of antioxidants and oxidants in cells or tissues is disrupted. Oxidative signaling is reported to protect the biological response in healthy cells by acting as a secondary messenger and playing a role in many cell signaling pathways. However, oxidative stress occurs when reactive oxygen species are formed excessively or when the concentration of antioxidants is low, resulting in oxidative injury. Oxidative stress generated by NOX is considered to be a secondary effect rather than the primary cause of disease; however, it can have a powerful effect on magnifying the resulting damage suffered as a consequence of a given injury. Oxidative stress has been reported to be involved in diseases, such as inflammatory disease; hypoglycemia; cardiovascular diseases, such as stroke; and neurodegenerative disease, including Parkinson’s disease and Alzheimer’s disease. It has been reported that, in these diseases, neurological outcomes are improved through treatment with antioxidants, post-injury. Based on these findings, we describe the mechanisms responsible for the generation of ROS due to NOX in vivo and the sequential neuronal death. We also describe how NOX activity is associated with neurogenesis after acute neurological illness.Oxygen -type glutamate receptor ,23. Exciironment .Free zinc is known to mediate NOX activation and exacerbate neuronal death. There is a high concentration of zinc in neurons . Since 1The main pathological elements of both types of stroke, ischemic and hemorrhagic stroke, are oxidative stress derived from an impaired supply of blood, oxygen, and glucose. In ischemic stroke, ROS are generated in a local hypoxic lesion, as anaerobic metabolism dominates. This process includes the free radical superoxide, primarily generated not from the uncoupling of nitric oxide synthase (NOS) but from NOX and mitochondrial enzymes. ROS also disrupt the blood–brain barrier, inducing cytokines, such as metalloprotease and also NF-kB via vascular endothelial growth factor (VEGF) expression. These inflammatory reactions also lead to the generation of ROS, worsening the penumbra region ,48. ThusDuring intracerebral hemorrhages (ICH) and subarachnoid hemorrhage (SAH), activation of NOX is detected. Immunoreactivity to the gp91phox subunit was found to be increased during both ICH and SAH models, especially NOX2 ,51. In aConsidering that NOX is essential for the production of ROS and leads to brain cell damage in various types of stroke, controlling the activation of NOX was thought to minimize neuronal damage from oxidative stress. NOX inhibition was experimentally achieved by either the implementation of NOX-related gene knockout mice or the administration of apocynin, a selective NADPH-oxidase inhibitor with IC50 of 10 μM . NumerouTraumatic brain injury not only includes primary mechanical injury but also secondary neuronal injuries in chronic stages. Primary neuronal injury results in inflammation spreading of cerebral damage, disruption of the BBB, and sequential neuronal damage. Under such circumstances, ROS is produced and deteriorates the viability of neurons. NADPH oxidase activation was observed 1 and 24–96 h after TBI, parallel to the rise of ROS. In a study examining only late time period (24 to 48 h) after TBI, it was confirmed that NOX2 activation in microglia significantly contributed to neuronal damage after TBI at 24 to 48 h, and NADPH oxidase activation increased ROS, as observed in the study. The likely cause would be the scavenging of ROS production by the administration of the NADPH oxidase inhibitor, apocynin . The proNOX inhibition has shown a neuroprotective effect after TBI in the hippocampal region. Our previous studies demonstrated this with the induced TBI model using mice and 24 h of apocynin injection (100 mg/kg). Detecting the oxidative stress with 4HNE staining showed that the inhibition of NOX led to an increased survival rate of pyramidal cells. The result was consistent with results of BBB disruption and microglial activation responsible for increased ROS  Table 1Table 1.Epilepsy can have genetic causes or result from acquired neurological injuries, such as a traumatic brain injury, infection, or stroke, in a process known as epileptogenesis. In genetic epilepsy, a variety of pathogenic variants in genes governing ion channel, mitochondrial, and endogenous antioxidant systems may predispose individuals to developing the disease. In previous studies, with diverse models to silence or overexpress the endogenous antioxidant response, involving agents, such as thioredoxin, oxidative stress was seen to be a crucial part of epileptogenesis. Epilepsy activates astrocytes and microglia, making them hypertrophic and reactive. Reactive glial cells release pro-inflammatory mediators, which, in turn, act on neurons, increasing the expression of redox-sensitive transcription factor activating protein -1 (AP-1) and NFκB, thereby activating NOX in microglia. Activation of NOX in microglia increases the production of extracellular peroxide via iNOS. These factors can affect neuroblasts or neurons that have recovered after epilepsy . In acquBoth reducing neuronal death and maintaining the integrity of the BBB are important features for neuroprotective agents in the context of oxidative stress. Notably, NOX inhibition can positively influence such variables . NOX inhThe cerebral cortex and hippocampus are the most vulnerable areas to hypoglycemic damage. The damage is driven not only by energy metabolic aspects but also by multiple synaptic changes, mainly arising from glutamate excitotoxicity. Hypoglycemic neuronal death also involves zinc-accumulation-associated damage. Hyperglycemia induces excess glutamate, aspartate, and zinc accumulation. Moreover, postsynaptic zinc accumulation results in reactive oxygen species (ROS) production by mitochondria or NADPH oxidase, leading to DNA damage and the activation of poly (ADP-ribose) polymerase-1 (PARP-1), mitochondrial permeability transition (MPT), and cell death ,68. AfteConsidering the pathophysiological connection between NOX and hypoglycemia, it is perhaps unsurprising that the inhibition of NOX alleviates the impact of hypoglycemia. The p47phox-deficient mice that lack NOX protein assembly showed less ROS production after hypoglycemia with ethidium fluorescence staining. The same study provided evidence that zinc chelation also ameliorates neuronal death. As the influx of excessive zinc from hypoglycemia activates NOX, NOX inhibition may have therapeutic usefulness in reducing cell death from ROS after hypoglycemia .The neurogenesis process is prominent in two parts of the adult brain: the olfactory region of the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampus . Neural All NSCs exist in the heterogeneous state, which undergoes both symmetric division  or asymmetric division (one mature to neuron). In both niches, NSCs migrate to certain brain regions and differentiate to immature neurons, showing hyperexcitability and increased susceptibility to synaptic inputs . The proThe stage of neurogenesis can be experimentally assessed with prominent biomarkers. Cells that are undergoing DNA replication can be detected with BrdU or H3-thymidine; BrdU-positive cells indicate neuron progenitors. Specifically, the M-phase of mitosis can be tracked by the green fluorescent protein (GFP) or Lac Z, and G1 to S or G2 to M by PCNA. Ki-67 is the marker for dividing cells. SOX gene families are thought of as strong markers for NSC proliferation. GFAP is shown in cells that have astrocytic traits, being an initial marker for neurogenesis as described above. Polysialic acid-neural cell adhesion molecules (PSA-NCAMs) are a strong marker for migration, being a detector in the late stage of the neurogenesis. A similar role can be observed for doublecortin (DCX), neuronal nuclei (NeuN), and proopiomelanocortin (POMC), as they signify immature neurons. NeuroD indicates differentiated cells during neurogenesis, which can be a form of identification of mitotic early neuronal lineage ,78,79,80Neural stem cells (NSCs) maintain proliferation and differentiation capacity in the brain. NSCs can also initiate self-repair mechanisms in response to neuronal damage. Several studies have confirmed that neural progenitor cells (NPCs) generated in the subventricular region (SVZ) and subgranular zone (SGZ) of the dentate gyrus of the brain migrate to the damaged brain region for neuronal repair. Migrated neuronal progenitor cells contribute to the recovery of cognitive abilities when brain damage occurs ,86,87,88In neuronal disorder studies, such as TBI, HG, GI, and seizure models, it was confirmed that NPCs migrate to the damaged area and differentiate into mature neurons. When single or prolonged neuronal damage occurred repeatedly, SVZ and DG were confirmed to demonstrate transiently increased neurogenesis ,91,92,93Post-stroke analysis has demonstrated that NOX inhibition can be effective in promoting neuronal survival. A study with NOX1 has quantified the magnitude of neurogenesis in AAV-induced NOX1-inhibited mice. There finding suggests that the survival rate of progenitor neurons after MCAO is high in NOX1-inhibited mice in the subventricular zone (SVZ) . ConsideAlthough epilepsy has shown neurodegenerative traits, it has been found that an epileptic seizure boosts hippocampal neurogenesis. However, this neurogenesis is not considered physiological neurogenesis in which neurons are formed and mature functionally but rather an ectopic response; newborn neurons in seizures are abnormal . AlthougThe programmed cell death (PCD) of neurons occurs in diverse stages during neuronal maturation periods. The apoptosis of migrating neuroblasts occurs with a lack of NMDA receptor expression and activities that increase neural correlation . With thThere are significant pieces of evidence that indicate that NOX activity is elevated after acute brain injury, and studies with NOX knockout animals and NOX inhibitors support the therapeutic possibility of targeting NOX subunits. Balancing NOX activity has also been shown to improve neurogenesis after acute brain injury. However, there remain many challenges ahead for the discovery of effective and NOX-specific targeting that would yield benefits in acute brain injury while minimizing negative side effects and generating a healthy neuronal micro-environment for ongoing maintenance neurogenesis. It is also noteworthy that NOX inhibitors, specifically NOX1 and NOX 4 selective inhibitors, have shown its safety and efficacy in reducing inflammatory markers in clinical trials. As such, pharmacological inhibition of a specific NOX for each neurological disease context may provide a safe and effective therapeutic potential in clinical setting as oxidative stress and inflammatory cascades are denominating, a feature of most acute brain injuries. While addressing these challenges remains a work in progress, the continued advancement of technology and research effort offers hope that the realization of this goal may be achievable in the near future in neurological diseases with unmet medical needs.


The elusive diversity of neurons puzzled neuroscientists since discovering the first nerve cells in the 1830s. Quantitative information about neuronal diversity began to flow from the middle of the twentieth century. At that time, microelectrode and histochemical tools were applied to vertebrate and invertebrate preparations. Simpler nervous systems of some gastropod mollusks, annelids, and nematodes revealed identified neurons with defined transmitter specificity and functions. Early systematic studies pointed out that most of the neurons composing their nervous systems might be unique genomics and transcriptomics, the astonishing diversity of neuronal cell types exceeds any imagination Moroz, . The mos Kandel, , aiming neurons evolved fromgenetically differentsecretory cells. The evolutionary view of neuronal evolution can be summarized as follows. Each of these populations of secretory cells could use chemically distinct transmitter(s) and different (distant) receptors for communications in early neural systems, where synapses are not required. Ancestral diversity of secretory cell types (=secretory phenotypes) has been preserved over 500+ million years of biological evolution, forming lineages of homologous neurons across phyla. Thus, neurons are different because they have different genealogies. Subsequent functional “demands” and specifications could further tune these different ancestral neurosecretory phenotypes. In other words, the traditional one-root genealogy of neurons was transformed into multiple genealogies or a net of phyletic cell/neuronal lineages, as schematically presented in In 1968–1974 these questions were asked from an evolutionary standpoint, and Dmitry Sakharov had proposed the hypothesis of neuronal polygeny (=multiple origins of neurons)  over many millions of years.The polygeny hypothesis stated that the transmitter-based primordial diversity of chemical signaling networks and mechanisms is the cornerstone for any nervous system organization. It was also suggested that neurons could evolve from heterogenous secretory cells in different tissues, or embryonic layers. In other words, neurons could evolve more than once within early precursors of ectoderm, endoderm, or mesoderm and/or other tissues. The presence of many transmitters in extant nervous systems reflects the complex chemical organization or The polygeny hypothesis also proposed the criteria and predicted neuronal homologs across taxa or within evolutionary different cell type lineages using modern terms.the existence of conservative neuronal cell types separated by million years of divergent evolution. Again, gastropod mollusks were used as reference species  History of the idea to trace the origins of neurons from secretory cells. (2) Postulates that substantiate different evolutionary scenarios for neuronal evolution. (3) Prospectives of multi-transmitter brain organization and transmitter-dependent behaviors essential to understanding the grammar of neural systems. The elusive chemical syntax of neural ensembles can explain brain operations' emerging properties, eventually leading to basal cognition different secretory cells that used multiple transmitters to communicate andintegrate behaviorswithout synapses. From this starting point, the evolutionary innovations multiply. Nerveless Placozoa and Porifera are two animal lineages that preserved such ancestral intercellular communications and, likely, the integration of behavior in a “pure” non-synaptic form peptides in early developmental control  category. The genetic category is referred to the scenario that all neurons are derived from the same ancestral cell lineage. Therefore, all neural systems and neuronal cell types are homologous because of their shared genetic ancestry. Alternatively, the functional category signifies examples of convergent evolution when similar chemical and physiological constraints resulted in similar neuronal phenotypes. This view does not prevent establishing evolutionary lineages of homologous cell types within particular taxonomical units such as classes, subclasses, orders, families, and genera. The cell-lineage-specific homologies across phyla are still a challenge  . Alexey Zakhvatkin (1906–1950) originally proposed this hypothesis (https://www.si.edu/object/siris_sil_363532), which obtained additional evidence  were preserved in extant nervous systems as decedents of early cell types. Most invertebrate ganglia, neural “circuits” or neural ensembles are composed of different cell lineages with distinct secretory phenotypes and evolutionary histories. I predict the reconstruction of hundreds of genealogies for metazoan secretory cells and neurons in particular. Neurons might evolve from ectodermal, entodermal, and mesodermal-type derivatives. See illustrative examples from the sea urchin  in early animals. As a result, the tightly coupled integrative transmitter systems (secretory phenotypes) are evolutionary conservative. Thus, the transmitter specificity can be instrumental in deciphering the homologous behaviors (=transmitter-induced motor outputs and behaviors). For example, serotonin acts as the integrator of behavioral [feeding] arousal in annelids . In contrast, dopamine is easily oxidized with several potentially toxic products (often leading to neurodegeneration).By acting within synaptic clefts and beyond, the transmitters are ds Lent,  and mollds Lent,  and manyds Lent, . Serotonds Lent, , but themore than 20 small and 100+peptide transmitters in nearly every nervous system, their chemical balances provide unprecedented opportunities for evolutionary innovations, behavioral controls, and behavioral choice—all uniquely realized in different animal groups. Thus, “transmitters made nervous system”  and short peptides were the first transmitters or co-transmitters. Multi-transmitter chemical wiring and integration were imperative both in Precambrian metazoans and the present-day animals. Some parasitic groups might have a reduced set of their neurotransmitters because of secondary simplification . Similarly, the ideal classification of cell types can predict their functional features and constraints transmitters and many thousands of novel neuropeptides. The secretory organelles are highly conserved across eukaryotes. The recent comparative study on the choanoflagellates (the sister group to Metazoa) clearly illustrated a polarized localization of putative but quite diverse secretory vesicles in two model species Salpingoeca rosetta and Monosiga brevicollis transmitters and synapses? Both functional  and evolutionary constraints  have to be considered. The chemical/secretory organization and relationships among the digestive, immune and neural cell types are unknown for basal metazoans.chemoconnectome is defined as an entire set of neurotransmitters, neuromodulators, neuropeptides, and receptors supporting chemical transmission in an animal  is the Frontier in cell, developmental, and evolutionary biology as well as biomedicine.A Multiplexed nanotools (Farsi et al., The combinatorial power of chemical interactions is enormous, but constraints of neurotransmitters signaling also exist, observed in well-defined phenomena of transmitter-dependent behaviors (Dyakonova and Sakharov, Gifts of Nature to neuroscientists and humankind. We are only getting the first surprises from these gifts. We are only starting to taste novel fundamental insights and paradigm shifts in this endeavor. It might be controversial, but the shortcut to better understanding our brains and neurological disorders and regenerative medicine of the future is studying small creatures in the world ocean. Admittedly, not all marine creatures can be brought to the lab and cultured. But we now have the capacity to bring labs to the sea (Moroz, I would conclude that the current biodiversity of species with the astonishing diversity of secretory and signaling mechanisms, neurons and synapses, neural and alternative integrative systems are true a Moroz,  and expaThe author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


With estimated numbers of 1–10 per mL of blood, circulating tumor cells (CTCs) are extremely rare compared to white (a few million) or red (billions) blood cells. Given their critical role in metastasis, CTCs have enormous potential as a biomarker for cancer diagnosis, prognosis, and monitoring of treatment response. There are now efforts to characterize CTCs more precisely through molecular and functional analysis, expanding the CTC effort from one of diagnosis and prognosis to now include the use of CTCs to specifically target cancers and discover therapeutic solutions, establishing CTCs as critical in precision medicine. This article summarizes current knowledge about CTC isolation technologies and discusses the translational benefits of different types of downstream analysis approaches, including single-CTC analysis, ex vivo expansion of CTCs, and characterization of CTC-associated cells.Circulating tumor cells (CTCs) have been recognized as a major contributor to distant metastasis. Their unique role as metastatic seeds renders them a potential marker in the circulation for early cancer diagnosis and prognosis as well as monitoring of therapeutic response. In the past decade, researchers mainly focused on the development of isolation techniques for improving the recovery rate and purity of CTCs. These developed techniques have significantly increased the detection sensitivity and enumeration accuracy of CTCs. Currently, significant efforts have been made toward comprehensive molecular characterization, ex vivo expansion of CTCs, and understanding the interactions between CTCs and their associated cells  in the circulation. In this review, we briefly summarize existing CTC isolation technologies and specifically focus on advances in downstream analysis of CTCs and their potential applications in precision medicine. We also discuss the current challenges and future opportunities in their clinical utilization. Cancer metastasis, the process of tumor cells spreading from a primary tumor to distant organs, is the primary cause of cancer morbidity and mortality, which is responsible for about 90% of cancer-related deaths . CirculaIn the following sections, we categorize existing CTC isolation technologies based on their distinct methods for isolating CTCs from other blood cells. We also describe the advances in downstream analysis of CTCs and their potential applications, such as single-cell analysis, CTC biobank, and CTC-associated cells in the blood. Finally, we address the current challenges and future opportunities in their clinical utilization.In the past decade, we have witnessed remarkable improvements in the efficiency and accuracy of isolation of CTCs from peripheral blood samples. Several CTC isolation technologies are now commercially available and can overcome previous limitations, such as the rarity of CTCs in blood samples and the heterogeneity of CTCs. We summarize current techniques categorized by their use of CTCs’ physical properties , biological properties based on tumor-specific markers expressed in the membrane of CTCs, and a combination of physical and biological properties using microfluidic devices .®)  to separate peripheral blood mononuclear cells and CTCs from whole blood [® system  combines the density gradient centrifugation system and a porous barrier that increases the depletion of mononuclear cells [The differences in density of the diverse blood components have led researchers to use the density centrifugation method  [® system  are two commercially available systems for isolation and capture of CTCs onto a membrane for downstream analysis applications [® technology , a microfluidic-based platform can capture and harvest CTCs from the blood using a filtration cassette, which is based on cell size and deformability [The first microfiltration system for CTC isolation was developed more than 50 years ago . However France)  and the ications . Recentlications ,23. Notaications . The Parmability ,25. Staimability . Additio® FX1 automated system , a label-free CTC enrichment that uses a microfluidic principle with the inertial migration of particles and a secondary flow in curvilinear channels, which allows the CTChip® FR  to separate the larger cancer cells from the blood cells [® technology  leverages differences in the dielectric properties of the cell, capturing cancer cells with positive DEP [Since filtration-based methods may cause mechanical damage to CTCs, thus affecting further downstream analysis, several contactless separation methods have been developed to avoid the damage to CTC viabilities during the enrichment process. The VTX-1 Liquid Biopsy System  uses a microfluidic approach that does not use any antibodies or microfilters to capture cells; instead, it uses two types of forces: a shear-gradient lift force and a wall-effect lift force that directs particles away from the wall of the microfluidic channels where the blood is injected. The cells are passively separated by size through the application of inertial forces in the microfluidic device that affect the cells’ positioning within the flow channel. In addition, inertial microfluidics generate microvortices that trap the larger tumor cells while allowing the smaller blood cells to pass. The tumor cells are trapped in the microfluidic device until released from the chip by lowering the flow rate . Anotherod cells . The dieod cells . The septive DEP . Anothertive DEP . Anothertive DEP .® system technology , the first FDA-cleared technology for CTC detection for metastatic breast, prostate, and colon–rectal cancer. The system is based on the positive enrichment of CTCs by immunoaffinity, using nanoparticles with antibodies targeting the epithelial cell adhesion molecule (EpCAM) antigens for CTC capture. After enrichment, fluorescent reagents are added for identification and enumeration of CTCs of epithelial origin that exhibit the phenotypes EpCAM positive; cytokeratins 8, 18, and 19 positive; and CD45 negative, a marker specific to leukocytes. A DNA stain, DAPI  fluorescence, is also added to identify the nuclei of both CTCs and leukocytes. The limitation of this technique is its dependence on the expression of EpCAM [The isolation and capture of CTCs based on biological characteristics of those heterogeneous cells utilize different biological markers expressed in the cells  18,19,2,60,61,62of EpCAM .Another positive enrichment method, AdnaTest , uses a combination of three different antibodies that bind to different antigens. This method detects gene transcripts of tumor markers EpCAM, the transmembrane mucin 1 (MUC-1) protein, and the human epidermal growth factor receptor 2 (HER2) using a multiplex reverse transcription polymerase chain reaction (RT-PCR) . The metThe magnetic-activated cell sorting system (MACS) is a method of separation of different cell populations depending on their surface antigens or cluster of differentiation (CD) molecules . The CTCMicrofluidic-based enrichment technologies have been explored for CTC detection methods. The first microfluidic device or affinity-based microchip system is the CTC-chip, which consisted of an arrangement of microposts where the CTCs are captured as the whole blood is pushed over the surface of the chip. The platform captures and analyzes the target cells by using antibody-based separation, with antibodies such as anti-EpCAM . The sec® , uses the technology of random distribution of posts to disrupt the laminar flow. It uses a Biocept® antibody capture cocktail  against tumor-associated antigens from cancer cells of both epithelial and mesenchymal phenotype, as well as cancer stem cells [® platform  is a high throughput CTC flow cell-associated automated system that continuously flows liquid through a laminar flow cell using a programmable logic-controlled pump and a pipetting arm. The system is composed of a three-layer sheath flow for a positive selection of the CTC population from whole blood using antibody beads anti-EpCAM, anti-MelCAM (melanoma cell adhesion molecule), anti-HER2, anti-MUC-1, and anti-TROP2 or TACSTD2  [The geometrically enhanced differential immunocapture (GEDI) chip is a technique to isolate CTCs using size-dependent collision frequency that maximizes the CTC–wall interactions while minimizing the interactions of other blood cells and antibody-coated 3D posts. This technique can achieve high efficiency and high-purity capture as it allows for the combination of more than one antibody marker . Anotherem cells . The Isoem cells . The Liqducer 2) .The affinity-based microchip system, which consists of an arrangement of microposts, is a more complex microfluidic device system when compared to the surface-based microfluidic technology. An example of this technology is the microvortex-generating herringbone-chip (HB-chip), which applies a passive mixing of blood cells through the generation of microvortices to increase the interaction between the CTCs and the antibody-coated chip surface . Sheng aThe NanoVelcro CTC chip  is composed of an overlaid polydimethylsiloxane (PDMS)-based chaotic mixer, anti-EpCAM antibody-coated silicon nanowires (SiNW), and a multilayer chip holder that assemble the functional components to immobilize CTCs. The system engenders vertical flows of the blood sample with a considerable speed that enhances the contacts between CTCs and the capture substrate, preserving cell morphology ,78. AnotExpanding the variety of detection methods and the including markers that help to characterize cell populations at different phenotypic stages is crucial for improving cancer cell detection. The low abundance in blood and limited blood volume accessible from a cancer patient may represent a significant constraint on the detection of a particular cell population, such as the CTCs and their clusters. To address those limitations, nanotechnology approaches have been proposed to identify and isolate these cells. For example, with the ability to report a protein expression profile for CTCs as a function of surface marker expression, magnetic ranking cytometry (MagRC) has achieved a high level of sensitivity and resolution. The microfluidic chip used for MagRC contains 100 distinct zones with varied magnetic capture zones. An array of X-shaped structures generates regions of low velocity, and circular nickel micromagnets within the channel enhance a magnetic field that is applied externally, promoting efficient CTC capture . In addiRecent advances in nanomaterials and nanotechnology offer high sensitivity, specificity, and multiplexed measurement capacity in CTC isolation and detection ,84,85. F®  contains a Seldinger guidewire functionalized with EpCAM for capturing live CTCs when applied in the peripheral vein. This technique allows the flow of up to 1.5 L of blood, and the collected CTCs are characterized by immunocytochemical staining [+ CTCs can be directly detected by using in vivo flow cytometry (IVFC) in animal models [In addition to in vitro isolation of CTCs, several techniques and devices have been developed and show the potential for in vivo applications . GILUPI CellCollectorstaining . Recentlstaining  developel models . Moreovel models .CTC counts have shown to have a promising prognostic value in monitoring cancer progression and therapeutic response for advanced metastatic patients. The efficacy of using CTC counts as a robust independent biomarker in clinical settings, however, is still under investigation. The most important limitation is that there is no gold-standard CTC approach that is widely agreed upon in the CTC community. Since CTC isolations and enumerations are conducted in different laboratories using different techniques, they result in disparities in CTC quantification depending on the method of choice. Moreover, CTCs are extremely rare in early-stage cancer patients and in advanced metastatic patients. For these patients, employing CTC counts alone as a prognostic marker may introduce biases in the early prediction of the outcome. Other factors can also affect the baseline CTC count of cancer patients. Inflammatory breast cancer patients and small-cell lung cancer (SCLCL) patients with chronic obstructive pulmonary disease (COPD), for example, may have a greater chance of releasing tumor cells into the bloodstream than other types of cancer patients due to high vascularity and increased microvessel density ,95.Currently, CTC research is extending beyond enumeration for more accurate cancer diagnostics. Immense efforts have been devoted to a genotypic and phenotypic analysis of CTCs, such as molecular characterization, ex vivo expansion, and investigation of crosstalk between CTCs and their associated cells . The information obtained from these studies provides unprecedented insights into the metastatic process and allows us to explore prognostic biomarkers and therapeutic targets for cancer management .Identifying molecular defects associated with cancer progression and drug resistance is essential for the improvement of cancer diagnostics and therapeutics. Although molecular interrogation of CTCs holds great promise as a noninvasive “liquid biopsy” for real-time monitoring of the change of cancer drivers, it may also introduce a strong bias in the molecular analysis due to the extremely rare events of CTCs in the impure samples, even after an enrichment process. As a result of recent advances in technology , researchers are now able to perform in-depth molecular characterization of CTCs at the single-cell level, which provides more clinically useful information when studying CTC heterogeneity and improving early patient stratification . Of the In comparison with genomic analysis, transcriptome analysis provides more detailed information that allows us to precisely identify cancer drivers for diagnosis and therapeutic targets . AlthougIn addition to the investigation of DNA and RNA biomaterials, researchers are also investigating the important roles that microRNAs and epigenetic modification play in mediating gene expression . Thus, profiling microRNA and DNA methylation in CTCs can also make a valuable contribution to cancer prognostics ,116. UnlResearchers have widely used established cancer cell lines for studies of cancer pathological mechanisms and anticancer drug testing. Despite their significant role in cancer research, cancer cell lines do not completely mimic the original solid tumors . InsteadAlthough the establishment of cell lines from CTCs has been challenging due to the rarity of CTCs in blood, significant advances in viable CTC enrichment techniques and culture systems have been making it possible. In 2005, Alix-Panabieres et al. developed the EPISPOT assay method, which enables a short-term culture of CTCs ex vivo from cancer patients . In 2013Cancer drug screening in patient-derived tumor cells holds great promise for translational and personalized medicine. Developing a physiologically relevant cell culture system is critical to the advancement of accurate and efficient identification of drugs. Recently, researchers have developed a novel culture system, known as organoids, which can mimic many structural features and pathophysiological functions of tumors ,124,125.Circulating tumor microemboli (CTM), so-called CTC clusters, the heterogeneous multicellular clumps that are released from the primary tumor, have been shown to have higher metastatic potential than single CTCs in animal and clinical studies ,130. CTMThe tumor microenvironment is a complex ecological system where tumor cells, stromal cells, infiltrating immune cells, and extracellular matrix, as well as the blood and lymphatic vascular networks, together orchestrate cancer metastasis. In the primary tumor, tumor-associated macrophages (TAMs) have been well studied as critical metastatic effectors and demonstrated to stimulate angiogenesis and promote tumor cell proliferation, invasion, and intravasation . In blooTumor-associated stromal cells can also significantly contribute to the tumor microenvironment, acting as feeder cells to support tumor growth and development . These fInnovative approaches have dramatically expanded the CTC field in recent years. Although promising results have revealed the potential clinical value of CTCs, the utility of CTC tests in clinical settings still faces significant challenges. To achieve the same widespread clinical utility as other diagnostic tests, such as pregnancy or blood glucose tests, several limitations need to be overcome. Based on the current standard procedure of CTC tests, one of the challenges comes from CTC sampling where 7.5 mL of blood sample is withdrawn from venous blood vessels. Because CTCs are extremely rare in the blood, it is critical to consider whether the sample size (7.5 mL) accurately reflects the total of 5 L of blood in an adult human. The evidence has shown that increasing the sample volume from 7.5 mL to 30 mL significantly improves the CTC detection rate from 13% to 47%. This indicates that a large sample size or multiple blood drawing may be the essential requirement for CTC tests in clinical settings in the future . Another® system, using anti-EpCAM antibody-conjugated magnetic beads isolates CTCs followed by confirming CTCs using pan-cytokeratin (Pan-CKs). However, downregulation of epithelial markers on CTCs, such as EpCAM, is associated with epithelial–mesenchymal transition (EMT), a biologic progress where epithelial cells acquire a mesenchymal phenotype and thus become more aggressive and invasive. Thus, the CellSearch® system is unable to isolate EpCAM negative CTCs. As a result, the system underestimates the number of CTCs and only detects them in about 60% of metastatic breast cancer patients. Moreover, CK-negative CTCs can often be found in patients associated with poor prognosis. Therefore, there is an unmet need for the investigation of specific CTC biomarkers. The researchers have used cell surface vimentin, a well-known EMT marker, to isolate CTCs and showed that EMT CTCs correlate with disease progression or relapse of cancer patients [Technological advancements have led to the development of many innovative CTC assays that can significantly improve CTC detection; however, this variety has led to discordance in the CTC community due to methodological discrepancies. Therefore, the requirement of clinical validity in larger studies for these new CTC detection techniques is essential for future clinical utility. Other technical factors may also contribute to the limitations of CTC tests in clinical settings. For example, process time and procedure may cause damage to CTCs, resulting in variability and bias for data interpretation. Furthermore, accurate identification of CTCs remains an enormous challenge owing to tumor heterogeneity. Concerning the CellSearchpatients ,154. Notpatients . A multipatients ,158,159.patients . This fiEarly detection of cancer is critical for the reduction of cancer morbidity and mortality. CTCs have recently emerged as a surrogate marker, and screening for CTC has the potential not only for early cancer diagnosis but also for real-time monitoring of disease progression and therapeutic response. In addition to CTC enumeration, downstream analysis of CTCs provides valuable insight into mechanisms of cancer metastasis, which allows us to identify targets for the most effective pharmaceutical treatments. In the future, although utilization of CTCs in clinical practice still has many limitations that require a high level of clinical validity, a combination of CTCs with other liquid biopsy partners, such as cell-free DNA and exosomes as an alternative to traditional biopsies, will pave the road for early cancer diagnosis and personalized treatment.


The cyclic GMP‒AMP synthase (cGAS)‒stimulator of interferon genes (STING) signaling pathway senses the presence of cytosolic DNA and, in turn, triggers downstream signaling to induce the expression of inflammatory and type I interferon genes in immune cells. Whereas the innate immune function of the cGAS‒STING pathway is well studied over the past years, emerging evidence suggests that this signaling pathway may have additional functions beyond innate immune surveillance. Consistent with this notion, dysregulation of the cGAS‒STING signaling pathway in adipocytes, hepatocytes, and renal proximal tubule epithelial cells are associated with metabolic dysfunction, impaired energy homeostasis, and kidney diseases. In this review, we summarize current understanding of the cGAS‒STING pathway in several metabolic diseases such as obesity, insulin resistance, alcoholic and nonalcoholic fatty liver diseases, as well as acute kidney injury and chronic kidney disease. We also review the interaction between the cGAS‒STING pathway and lipid metabolism. Lastly, we discuss potential mechanisms by which cGAS‒STING signaling regulates metabolism and point toward future avenues of research targeting the cGAS‒STING pathway as possible means to treat common metabolic disorders. The cyclic GMP‒AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that plays a vital role in innate immune defense against pathogen infection or damage-induced host DNA aberrantly localized in the cytosol . Activathttps://www.proteinatlas.org/ENSG00000164430-CGAS/tissue). We and others also detected cGAS expression in several metabolism-related tissues in mice including the epidydimal adipose tissue, inguinal adipose tissue, brown adipose tissue, liver, heart, and kidney (https://www.proteinatlas.org/ENSG00000184584-TMEM173/tissue). High STING protein expression was also detected in a number of other nonimmune tissues such as the lung, rectum, pancreas, testis, prostate, urinary bladder, heart muscle, and adipose tissue, but little STING expression was detected in the liver, smooth muscle, or skeletal muscle. These results are consistent with the finding that human or mouse hepatocytes do not express STING , Kupffer cells (CD68+), and CD163+ macrophages , which is characterized by accumulation of excess fat in the liver of people who consume little or no alcohol, contributes to the development of nonalcoholic steatohepatitis (NASH) and potentially cirrhosis and hepatocellular carcinoma . Increasrophages . Transplrophages . NeverthIn addition to playing a role in nonalcoholic fatty liver disease (NAFLD) development, activation of the cGAS‒STING signaling pathway has also been shown to contribute to liver injury in alcohol-related liver disease (ALD) . The expDysregulation in cholesterol and fatty acid synthesis and/or their cellular uptake leads to various metabolic disorders such as NAFLD . The synAccumulated evidence reveals that lipid metabolism and type I IFN response are coregulated, which enables innate immune cells to coordinate with metabolic changes required for antiviral responses . In suppIn addition to cholesterol, perturbance of fatty acid metabolism by inhibiting fatty acid binding protein 5 (FABP5), a lipid chaperone that facilitates fatty acid uptake and intracellular lipid trafficking, also leads to the activation of cGAS‒STING-mediated type I IFN signaling . GeneticKidney, a mitochondria-enriched and metabolism-active organ, is the second highest consumer of molecular oxygen in the body at rest . Under rTfam-knockout mice , which are shown to be both type I IFN-responsive and type I IFN-productive cells . VSMCs cThough significant progress has been made over the past several years in understanding the role of the cGAS‒STING signaling pathway in cytosolic DNA sensing and host defense , emerginIncreasing evidence suggests a close and dynamic link between the immune and other biological systems in our body . The croRecent studies reveal that cGAS is localized not only in the cytosol but also at plasma membrane  and in tin vivo studies were done by using global cGAS or STING knockout mice, it remains to be determined as to how much of the observed phenotypes is due to a direct effect of cGAS or STING signaling in a given cell/tissue as opposed to a secondary effect. It is also unclear how the cGAS‒STING signaling pathway is specifically regulated in distinct cells or tissues. Furthermore, dysregulation of the cGAS‒STING pathway has been implicated in a spectrum of pathological conditions, but what is the connection between cGAS and/or STING dysregulation and metabolic dysfunction, cancer, autoimmune diseases, neurodegenerative disorders, and aging? It is likely that our knowledge will be greatly improved with the phenotype characterization of cell-specific cGAS or STING knockout animal models and with the development of more specific pharmacological agents targeting components in the cGAS‒STING signaling pathway. Understanding the tissue/cell-specific roles of the cGAS‒STING signaling pathway will provide novel and accurate insights into the mechanisms linking the immune and other biological systems in health and diseases.In conclusion, despite a great advancement on our understanding of the role of the cGAS‒STING signaling pathway in innate immunity, a number of outstanding questions remain to be addressed. Given that many of the This work was supported in part by grants from the National Natural Science Foundation of China  and Innovative Basic Science Awards of American Diabetes Association (1-19-IBS-147).Conflict of interest: none declared.