Viral infection is an indisputable causal factor for nearly 17% of all human cancers. However, the diversity and complexity of oncogenic mechanisms raises new questions as to the mechanistic role of viruses in cancer. Classical viral oncogenes have been identified for all tumor-associated viruses. These oncogenes can have multiple oncogenic activities that may or may not be utilized in a particular tumor cell. In addition, stochastic events, like viral mutation and integration, as well as heritable host susceptibilities and immune deficiencies are also implicated in tumorigenesis. A more contemporary view of tumor biology highlights the importance of evolutionary forces that select for phenotypes better adapted to a complex and changing environment. Given the challenges of prioritizing singular mechanistic causes, it may be necessary to integrate concepts from evolutionary theory and systems biology to better understand viral cancer-driving forces. Here, we propose that viral infection provides a biological “entropy” that increases genetic variation and phenotypic plasticity, accelerating the main driving forces of cancer cell evolution. Viruses can also influence the evolutionary selection criteria by altering the tumor microenvironment and immune signaling. Utilizing concepts from cancer cell evolution, population genetics, thermodynamics, and systems biology may provide new perspectives on viral oncogenesis and identify novel therapeutic strategies for treating viruses and cancer. Viruses have well-established causal roles in numerous human and animal cancers, collectively responsible for almost one fifth of all cancers , 2. ViraOncogenic viruses perturb numerous cellular pathways described as the hallmarks of cancers , 4. As eA major challenge in the field of viral oncology, and cancer biology in general, is the very large number of mechanisms and pathways that contribute to carcinogenesis. While early studies focused on one or a few viral oncogenes, we now know that viruses can promote cancers through a much greater diversity of mechanisms and pathways. Many of these mechanisms can be well-defined in the context of a particular tumor type or tissue environment. However, the diversity of mechanisms confounds the identification of single causal agent or event. Given the abundance of potential and actual mechanisms, a new challenge arises to both understand the impact of each oncogenic event and the accumulation of multiple distinct oncogenic events over time or in the context of a transient stress challenge. Here, we provide a brief overview of some classic mechanisms of viral oncogenesis, highlighting many from the human gammaherpesviruses, and argue that the multiplicity of these mechanisms makes sense only in the context of cancer cell evolution and systems biology.Viruses have been shown to induce tumor formation through numerous and diverse mechanisms . From thPioneering studies identified a few cellular targets universally exploited by tumor viruses, such as p53 DNA damage surveillance and Rb cell cycle control. However, it is now known that subversion of these targets are not sufficient for viral tumorigenesis, and that additional and alternative host proteins and pathways are targeted by viral oncogenes . For exain vitro  and utilize alternative metabolites, such as glutamine and serine for energy production and macromolecular biosynthesis . OncogenViral-infected cells and associated tumors thrive in harsh microenvironments that reinforce viral-infected and cancer cell selection, requiring Warburg metabolism and adaptation to low oxygen and acidification , 65. VarIn Darwinian terms, viruses and cancer are most limited by the predatory function of the immune system. Viral associated cancers are particularly adept at modulating immune surveillance, and are most virulent in immunosuppressed conditions, such as HIV-AIDS and solid-organ transplants. HPV and EBV associated tumors upregulate T-cell checkpoint proteins PD-L1 and PD-L2, as well as the CTLA-4 immunosuppressive pathways . MultiplPerturbations in transcription factor and gene regulatory networks are also hallmark changes in cancer. Viral immediate-early genes and oncogenes frequently target transcription factors and transcription factor networks that are fundamental to host cell differentiation and identity. E6, E7, and T-antigens are known to interact with cellular transcription factors, such as p53 and the Rb-family complexes. EBV and KSHV encode numerous other nuclear factors that alter transcription control, and these viral factors have been implicated in viral carcinogenesis. All six EBV-encoded EBNAs interact with host transcriptional regulators to perturb regulatory networks in distinct and complex ways. EBNA2 can interaction with B-cell regulatory factors RBPJ, EBF1, RUNX1, and PU.1 to affect cooperative DNA binding site selection  as well Epigenetic modifications represent an important mode of adaptive and heritable gene regulation. Persistent viral infection can impact host epigenomes in diverse ways. DNA infection in the nucleus can alter the DNA methylation patterns, frequently resulting in hypermethylation of viral and cellular genes . Host geViruses can also induce changes in host chromosome conformation leading to the rewiring of gene regulatory circuits. EBNA-LP, EBNA2, and EBNA3C have been implicated in the reorganization of DNA regulatory loops to form super-enhancers regulating cellular oncogenes, such as c-myc, to drive resting B-lymphocytes into proliferating and immortalized lymphoblastoid cells , 114. EBAll known human tumor viruses persist, in one form or another, at the site of tumor formation. Tumor viruses can persist as chronic infections (HCV), nuclear episomes  or integrated genomes in viral-associated tumor cells (HTLV-1). HCV is an unusual oncovirus in that it does not infect the cancer cell, but its long-term persistence causes inflammation conducive to cancer cell emergence. In contrast, episomal DNA tumor viruses, like EBV, KSHV and HPV have viral-specific programs dedicated to viral genome persistence in a dividing cell that can serve as a clonal outgrowth in cancer –121. TheChang and Moore proposed that viral cancers arise due to aberrations in the normal productive life cycle, including genetic mutations and integrations that disrupt normal viral gene expression . ConsistTumor cell heterogeneity is a central hallmark of cancer –134. VirViruses can be highly heterogenous and mutate during the course of infection to increase carcinogenic risk. High and low risk subtypes of HPV may be considered a form of species heterogeneity, while integrations and deletions can be a source of mutational variation , 137. TuTemporal history of viral infection may also contribute to tumor heterogeneity . Viral cHost genetic variation, whether inherited or acquired through somatic mutation, also contribute to the risk of viral cancers. Variations in the HLA locus correlate with risk of viral cancers, suggesting that presentation of viral antigens plays a key role in immune resistance to viral-driven cancers. Genetic analysis of NPC susceptibility revealed risk loci at hTERT, CDKN2A/B, MECOM, and TNFRSF19, all of which have known roles in oncogenic pathways. Other susceptibility pathways have been linked to Notch signaling, magnesium transport (NIPAL1), EBV entry into epithelial cells (ITGB6), modulation of apoptosis , cAMP signaling, or DNA repair  . InheritOther aberrations also contribute to variations in viral-host interaction. Rare tumors of atypical tissue types, such as EBV leiomyosarcomas, NK-T cell lymphoma, peripheral T-cell lymphoma  and pulmStage specific effects on viral oncogenesis are observed for most oncoviruses. HPV has been shown to have different patterns of infection, integration, and gene expression at different stages of viral-associated cancers . SimilarOncogenic viruses increase the number of possible ways a cell can propagate, survive and achieve oncogenic transformation. In terms of Darwinian evolutionary dynamics and population genetics, virus infection increases the phenotypic diversity and fitness of the population. A more heterogeneous population has a fitness advantage by adapting more rapidly to changing and stressed environments. To borrow from systems biology, a stable phenotype requires a stable gene regulatory network (GRN). GRNs are considered a thermodynamic “attractor” state  Figure . GRNs arIn thermodynamics and statistical mechanics, terms like entropy were developed to explain the behavior of complex systems with excessively large numbers (ensembles) of microstates. In this respect, the term entropy can be used to describe the number of microstates of a complex biological system, such as the gene regulatory interactions in a viral infected tumor cell. While we can not provide a rigorous definition of biological entropy, we do suggest that viruses increase the number of possible microstates available to the host cell, and therefore may be considered a form of “genetic entropy.” In the most simplistic terms, genetic entropy may be the ability to reconfigure the genome and its programmed processes. Viruses reconfigure genomes, gene expression programs, and biochemical pathways. We further suggest that this be considered in terms of Shannon information theory, where viral genomes may be considered a source of signal noise enabling the freedom to find a lower energy state, or alternative GRN, such as the oncogenic state. Viruses can increase signal noise and alternative outputs for cellular developmental programs, and this enables transcriptional plasticity and phenotype heterogeneity associated with tumorigenesis. Efforts to quantify cellular information and thermodynamic entropy may be useful for understanding the emergence of the cancer phenotype. In this regard, virus infection may be considered an entropic driving force for cancer .The diversity of oncogenic mechanisms and the plasticity of cancer cells raise enormous challenges in developing precision therapies. For most cancers, early detection provides the best opportunity for effective treatment. Viral cancers have the advantage of having viral-specific targets and biomarkers. For example, early-stage NPC can be predicted from cell-free EBV DNA in plasma  and EBV-Viruses are thought to be simple biological systems, yet their contributions to cancer can be fiendishly complex. If we return to the question of why some viruses cause cancers but not closely related others, we have only a partial answer. Among the common features, is that of long-term persistence of tumor virus in localized tissue compartments. Another common feature, as highlighted by Chang and Moore, is the frozen accident of the defective virus entering the wrong cell type, or cell with precancerous mutation, or acquired mutation in host or virus that blocks the natural infection and immune clearance. Host and virus genetic variations can be susceptibility factors that enable viral oncogenesis. One additional feature is the plasticity provided by chronic virus infection, and how that effects the survival options for infected tumor cells and tumor fields. Viruses co-opt and perturb numerous cellular pathways implicated in cancer. These perturbations may occur at different times  and in different subpopulations  and may be replaced by cellular oncogenic drivers at different stages of tumor evolution (interchangeability). Viral-specific cancer mechanisms may have unique features and provide new insights into cancer biology and genetic plasticity. Despite this complexity, viral cancers may be considered low hanging fruit for cancer therapeutic intervention. Prevention of virus infection and virus-specific inhibitors have been shown to diminish cancer risk, and immune targeting of viral proteins show clinical promise. Deeper understanding of the basic mechanisms driving cancer cell evolution may also be required for more effective intervention.Precision medicine requires discrete knowledge of the causal factors in disease. Identification of the specific drivers and treatment with selective drugs for each driver pathway is a reasonable, rational, and reductionist approach to cancer therapy. Viral cancers are likely to have different vulnerabilities than their non-viral counterparts, including mechanisms driving cellular plasticity and evolvability. Studying viral cancers may also help us to solve some of the key questions in cancer biology. What are the rate-limiting steps in cancer evolution? What are the most vulnerable nodes of a gene regulatory network for a particular type of cancer and how can we dampen genetic and environmental noise to reduce cancer cell plasticity and evolvability? Can we reverse tissue microenvironment conditions that preferentially select for cancer cell evolution? Ultimately, understanding the evolutionary and thermodynamic driving forces of virus infection and carcinogenesis will provide a more coherent conceptual framework for research and new avenues for therapy.


A recurrent laryngeal nerve (RLN) injury resulting in vocal fold paralysis and dysphonia remains a major source of morbidity after thyroid and parathyroid surgeries. Intraoperative neural monitoring (IONM) is increasingly accepted as an adjunct to the standard practice of visual RLN identification. Endotracheal tube (ET) surface recording electrode systems are now widely used for IONM; however, the major limitation of the clinical use of ET-based surface electrodes is the need to maintain constant contact between the electrodes and vocal folds during surgery to obtain a high-quality recording. An ET that is malpositioned during intubation or displaced during surgical manipulation can cause a false decrease or loss of electromyography (EMG) signal. Since it may be difficult to distinguish from an EMG change caused by a true RLN injury, a false loss or decrease in EMG signal may contribute to inappropriate surgical decision making. Therefore, researchers have investigated alternative electrode systems that circumvent common causes of poor accuracy in ET-based neuromonitoring. Recent experimental and clinical studies have confirmed the hypothesis that needle or adhesive surface recording electrodes attached to the thyroid cartilage (transcartilage and percutaneous recording) or attached to the overlying neck skin (transcutaneous recording) can provide functionality similar to that of ET-based electrodes, and these recording methods enable access to the EMG response of the vocalis muscle that originates from the inner surface of the thyroid cartilage. Studies also indicate that, during surgical manipulation of the trachea, transcartilage, percutaneous, and transcutaneous anterior laryngeal (AL) recording electrodes could be more stable than ET-based surface electrodes and could be equally accurate in depicting RLN stress during IONM. These findings show that these AL electrodes have potential applications in future designs of recording electrodes and support the use of IONM as a high-quality quantitative tool in thyroid and parathyroid surgery. This article reviews the major recent developments of newly emerging transcartilage, percutaneous, and transcutaneous AL recording techniques used in IONM and evaluates their contribution to improved voice outcomes in modern thyroid surgery. Although thyroid surgery is among the most common and safe interventions in endocrine surgery, the risk of complications is still evitable due to the unique anatomical structure and physiological function of the thyroid gland . RecurreCurrently, EMG recording during neuromonitored thyroid surgery is almost always performed using endotracheal tube (ET) surface electrodes placed in contact with vocal folds during intubation for general anesthesia because of the advantages including their noninvasiveness, wide commercial availability, and capacity to monitor larger areas of the target muscle , 5. Howe. These studies have also demonstrated that transcutaneous or transcartilage anterior laryngeal (AL) recording electrodes are as accurate as ET-based surface electrodes in depicting RLN stress during IONM. However, transcutaneous or transcartilage AL recording electrodes could be more stable than ET-based surface electrodes during surgical manipulation on the trachea. These findings indicate that AL electrodes have potential applications in future designs of recording electrodes and support the use of IONM as a high-quality quantitative tool in thyroid and parathyroid surgery. This article reviews recent studies of new and emerging transcutaneous or transcartilage AL recording techniques used during IONM and compares these techniques in terms of their contribution to improved voice outcomes after modern thyroid surgery.To avoid the time-consuming processes of verifying and readjusting the ET position during neuromonitored thyroid surgery and to minimize the safety hazard of signal inconsistency and instability. Regarding the basis of anatomy, there are many innovative hypotheses and possible solutions including novel electrodes designs have been proposed. For example, many experimental –18 and c) , surface recording electrodes placed on the outer surface of TC should enable access to EMG signals evoked in vocal fold muscles during IONM  . To test)  performe) . Zhao et)  investigSimilarly, Zhao et al.  performeIn a prospective clinical cohort study of 25 patients, Van Slycke et al.  affixed In pediatric patients, anatomical characteristics, e.g., smaller RLN diameter compared to adults, can make IONM challenging. One consensus statement suggested that IONM is beneficial in pediatric patients with a bulky thyroid and lymph node disease . Since t in which pairs of insulated needle electrodes  were inserted percutaneously into the TC perichondrium to within 5 mm of the tip. The four most widely used remote thyroidectomy techniques  were evaluated in animal experiments and in their initial case series. They concluded that percutaneous TC recording technique is feasible and can be modified according to the approach used for remote access. This technique reduces interruption of the surgical procedure while still providing reliable EMG signals  attached to the anterior neck skin. In their experiments, ET electrodes and neck adhesive skin electrodes successfully recorded typical evoked laryngeal EMG waveforms from RLNs and VNs under 1 mA stimulation. Additionally, both electrode types accurately detected adverse EMG events under experimentally induced RLN traction stress. Under experimentally induced tracheal displacement, however, EMG signals obtained by ET electrodes widely varied whereas EMG signals obtained by transcutaneous electrodes were stable   , Europe ®) , United ®) , and oth®)  provide The most important issue when IONM is performed using an ETT as the recording side is that IONM has a high negative predictive value (92%-100%) but a relatively low and variable positive predictive value (10%-90%) . The IONThis is the first comprehensive review in the literature of IONM with transcartilage , 27, 28,Whereas these novel techniques are apparently practicable and have no major disadvantages in most clinical applications, disadvantages of transcartilage electrodes include the higher skin flap elevation required to expose the TC . FinallyAlthough percutaneous AL electrodes are applicable in small incision thyroid and parathyroid surgery and in remote thyroidectomy, insertion of needle electrodes into the TC should be performed with extreme caution to avoid inadvertent injury to the cricothyroid muscle or EBSLN, which can cause muscular hematoma or scar tissue formation and subsequent alterations in vocal pitch. Additionally, percutaneous needle recording electrodes can inadvertently record far field potentials. Therefore, the recommended practices are confirming the stimulation site and comparing corresponding VN, RLN, or EBSLN waveforms, and adjusting the current intensity to minimize recording of far field potentials. Finally, excessive skin retraction or the use of skin retraction for counter-traction during remote thyroidectomy  could caAlthough transcutaneous AL recording in IONM has proven feasible and reliable, further technical refinement is needed to address some technical flaws. The skin electrode could be affected by patient characteristics such as obese or short neck, degree of subplatysmal flap elevation, and large tumor size. During surgery, subplatysmal flap and retraction of the strap muscle during dissection of the thyroid upper pole may hinder neural signal transmission. The EMG signals actually recorded after upper pole dissection might also be diminished . TherefoIn summary, this review of the recent literature regarding the feasibility, stability, safety, and efficiency of new and emerging techniques for obtaining transcartilage, percutaneous, or transcutaneous AL recordings during IONM found that recently developed techniques increase the potential applications of IONM as a high-quality quantitative tool in thyroid and parathyroid surgery. However, this review also revealed that most clinical studies have enrolled a relatively small number of patients. Therefore, future prospective studies in larger populations are needed to explore and verify the applicability and benefits of these techniques. Several novel techniques that can further improve AL recordings have been recently reported in the literature. One study uses a nanosheet-based microneedle for EMG recording, which minimizes the risk of laryngeal injury caused by needle insertion . InsteadIn the current era in which quality of life is increasingly included as a surgical outcome measure, voice outcome after thyroidectomy is an important consideration for both patients and any clinician involved in managing such patients. Several methods and technologies are being developed to overcome the limitations and disadvantages of the current IONM system that relies on EMG ET recording. This article reviews major recent developments and progress in new and emerging transcartilage, percutaneous, and transcutaneous AL recording techniques used during IONM. In comparison with the conventional ET recording method, many experimental and clinical studies have shown that these up-to-date AL recording methods have advantages of lower invasiveness, better EMG quality and stability, and avoidance of time-consuming disturbances for ET position verification. In addition, these methods are not only feasible for routine ordinary thyroid and parathyroid surgery, but also practical during thyroid or parathyroid surgery with a small incision, remote access, and in pediatric operations. Additional advantages of AL recordings include a cost-effective and surgeon-friendly set-up, allow for rapid implementation when an unexpected need, and the dislodged electrodes can be easily identified and managed intraoperatively. Although there are still some limitations of each recording method, we believe the continuous practical application and implementation of these developments will further optimize IONM to the ultimate benefit of thyroid surgery patients.CL, TY-H, CW-W, and GD conceived and designed the study. Administrative support was obtained by LF-W, HY-T, and YC-L. Provision of study materials by JW, L-PC, H-YT, Y-CL had collected and assembled the data. Data analysis and interpretation was done by F-YC, T-YH, and CL. All authors contributed to the article and approved the submitted version.This study was supported by grants from Kaohsiung Medical University Hospital (KMUH109-9M44), Kaohsiung Municipal Siaogang Hospital/Kaohsiung Medical University Research Center grants (KMHK-DK (C)110009, I-109-04, H-109-05, I-108-02) and Ministry of Science and Technology , Taiwan.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Myc-driven tumorigenesis involves a non-transcriptional role for Myc in over-activating replicative Cdc45-MCM-GINS (CMG) helicases. Excessive stimulation of CMG helicases by Myc mismanages CMG function by diminishing the number of reserve CMGs necessary for fidelity of DNA replication and recovery from replicative stresses. One potential outcome of these events is the creation of DNA damage that alters genomic structure/function, thereby acting as a driver for tumorigenesis and tumor heterogeneity. Intriguingly, another potential outcome of this Myc-induced CMG helicase over-activation is the creation of a vulnerability in cancer whereby tumor cells specifically lack enough unused reserve CMG helicases to recover from fork-stalling drugs commonly used in chemotherapy. This review provides molecular and clinical support for this provocative hypothesis that excessive activation of CMG helicases by Myc may not only drive tumorigenesis, but also confer an exploitable “reserve CMG helicase vulnerability” that supports developing innovative CMG-focused therapeutic approaches for cancer management. Myc is involved in the conversion of MCM complexes into CMG helicases for DNA replication, but too much Myc over-activates CMG helicases. This reduces the number of unused Reserve CMGs necessary for recovery from replicative stresses, promoting tumorigenesis but also creating an exploitable CMG weakness in tumor versus normal cells. Whereas such drivers promote carcinogenesis due to their ability to stimulate proliferative capacity of tumor cells, other drivers of cancer include proteins/enzymes whose decreased function creates a tumor-conducive environment within cells. These tumor suppressors are also typically rendered less active through inactivation or loss. Examples of the latter include loss of DNA replication fidelity due to mutation of MutS homolog genes, abrogation of apoptosis due to Bcl2 mutations, or loss of function mutations in tumor suppressor genes Tp53 and RB1. Mutation or deregulated expression of these drivers of tumorigenesis can be detected in cancers using next generation sequencing (NGS), exome sequencing, or genome wide association studies (GWAS). In contrast to these drivers, the majority of genes are frequently characterized as “housekeeping” and rarely or never mutated, nor included in targeted sequencing panels clinically.The development of cancer is a multistep process involving, in part, the acquisition or inheritance of mutations in oncogenes that renders their oncoprotein products overactive due to regulatory changes, gain of function mutations, or overexpressed conditions. Tumors are often dependent on these deregulated oncoproteins for development and maintenance, leading to the conventional definition of these oncoproteins as drivers of tumorigenesis. Examples of oncogenic drivers include mutation or deregulated expression of genes such as ,2 Such elevated expression of CMG components in tumor tissue is often referred to as “overexpressed,” providing an argument that CMG helicases (like other overexpressed cell cycle regulating proteins) might be drivers of cancer growth and thus suitable for the development of drugs that can inhibit CMG function for cancer treatment .Emerging evidence suggests that the replicative CMG  helicase may also be a driver of tumorigenesis despite it being in this last category of never-mutated genes. Many studies have found that tumor tissue and biopsy samples display elevated expression of CMG components relative to non-tumor tissue. in ref. ). Howevewww.cbioportal.org; accessed August 2, 2019) reveals that none of the 11 genes coding for CMG subunits contain hotspot mutations or amplifications beyond a basal background of overall genomic mutation rates. Collectively, these observations would argue that the CMG helicase and its components cannot be easily defined as overexpressed or mutated drivers of carcinogenesis as is often the case for many oncogenic proteins. However, understanding the biochemistry and cell biology of CMG helicase assembly and activation, and how this process is mismanaged by oncoproteins such as c-Myc and Cyclin E, produces a situation where the CMG helicase may well be a driver of carcinogenesis that is suitable for drug development and clinical intervention efforts.Consistent with CMGs not necessarily being overexpressed drivers of cancers, NGS, GWAS, and exome sequencing approaches tend not to identify alterations in CMG expression or gene integrity associated with human cancers, indicating that the genes coding for CMG subunits are not amplified or mutated in human cancers. For example, a search of more than 42 000 human tumor samples in cBioPortal , and a GINS tetramer (Go-Ichi-Ni-San) comprising Sld5 and Psf1–3,7,9 .,10,11,13 This serves two purposes. First, the MCMs mark chromosomes that have not yet replicated, and thus license such chromosomes for precisely one round of DNA replication.,14 Second, a subset of the MCMs that have licensed non-replicated chromosomal regions will become sites of future origins of DNA replication.,16 At Stage-2, which occurs in lateG1 or at the G1-S transition, Cdc45 and GINS are recruited to this subset of MCMs to form CMG helicases (as a pair) that will function in initiation and bidirectional elongation of DNA replication within replisomes.,6,7,17 As will be described in more detail below, emerging evidence has shown that in mammalian cells the c-Myc protein is required for and promotes the recruitment of Cdc45 and GINS to pre-existing MCM hexamers.–20 This role for c-Myc in CMG assembly and activation has the propensity to confer a driver function on CMGs during tumorigenesis, while simultaneously creating an exploitable vulnerability in CMG function in cancer cells that may provide a novel therapeutic target for cancer intervention.The replicative CMG helicase is an 11 subunit enzyme that catalyzes the melting of DNA during DNA replication and is required for resuming DNA replication during recovery from replicative stresses.f1–3,7,9 . Multipl2.2.1.,21 Excess unused MCM hexamers loaded throughout the genome (not seemingly converted to a CMG), while involved in licensing non-replicated DNA, are also known as dormant origins.,21,22 An interpretation of published results suggests that these reserve MCMs can become active CMG helicases during recovery from replicative stress.,16,23 Numerically, mammalian cells contain an ≈five- to tenfold excess of MCMs than are required to complete an S-phase.,8,16,23 Multiple studies have found that experimental depletion of the excess MCMs, while not blocking DNA replication itself, leads to sensitivity of cells to replicative stresses induced by exposure to replication fork-stalling drugs such as hydroxyurea and aphidicolin.,16,23 Predictably, further depletion beyond the reserve MCMs, reducing the MCMs minimally required for DNA replication to start and finish, halts S-phase progression.,16,23 It should be noted, though, that the actual fate of reserve MCMs at dormant origins in terms of their conversion to CMGs during ongoing DNA replication (but remaining inactive), or conversion only after replicative stress, has not been conclusively determined. Experimentally, a genomic chromatin-crosslinking, immunoprecipitation and sequencing (ChIP-seq) based approach analyzing multiple CMG subunits would be necessary to ascertain the degree to which MCMs are converted to CMGs before or during DNA replication, or after replicative stress, to give a clear picture of MCM-to-CMG dynamics. However, regardless of such molecular specifics, the excess reserve MCMs (or CMGs) are clearly necessary for efficient recovery from replicative stresses.A key for understanding how MCM/CMG mismanagement can perform a driver function for tumorigenesis, and as described later, an exploitable weakness in tumors comes from evidence that not all excessively loaded MCMs appear to be converted to CMGs during DNA replication.2.2. Importantly, these drugs are fork-slowing drugs that induce replicative stress on the tumor cells. Under these conditions of stalled replication forks, cells attempt to recover DNA replication (after any repairs) using the reserve MCMs as substrates to form a new CMG helicase and replicate remaining DNA regions.,16,23 The previously functioning CMGs are seemingly not used , and may be disassembled, although the fate of such previous CMGs remains poorly understood. Failure to utilize reserve MCMs for recovery yields DNA damage signaling responses and genomic instability.,21,23 Thus, during replicative stress, the dormant unused reserve MCMs are necessary for recovery of fork progression, thus ensuring fidelity of DNA replication and genomic stability.Of clinical relevance, reduction of the reserve complement of MCMs renders pancreatic ductal adenocarcinoma (PDAC) and colorectal carcinoma (CRC) cells sensitive to chemotherapeutic drugs commonly used to treat such cancers, in this case 5-fluorouracil, gemcitabine, and etoposide.2.3.Mcm4 gene.,24 The mutation in Mcm4, referred to as the Mcm4Chaos3 allele, does not result in a loss of CMG helicase enzymatic activity (as measured in vitro), but rather causes a destabilization of MCM hexamer binding to chromatin throughout the genome such that the entire cellular complement of MCM hexamers, including reserves, is functionally weakened.,24 Fibroblasts obtained from these mice show increased numbers of stalled replication forks, DNA damage signals, and activation of fork recovery events, even though DNA replication has not been perturbed by any external means such as fork inhibitors. Intriguingly, these problems in S-phase seem to be somewhat accepted by the cells, perhaps at a low level, and remain unfixed as cells enter M-phase. This leads to problems during chromosome segregation and an acquisition of genomic deficiencies. Although difficult to prove directly in these studies, it appears likely that these genomic problems promote tumorigenesis, as the mice carrying this mutant Mcm4 allele are tumor prone without additional tumor promoters, initiators, or interbreeding with other oncogene-carrying mice being needed. Depending on the mouse strain analyzed, breast adenocarcinomas, histiocytic sarcomas, or lymphomas arise.,24,25 In other studies, weakened dormant MCMs due to mutation of Mcm2 produce thymomas, lung carcinomas, or liver carcinomas depending on mouse strains assessed.,27 Thus, it may be that a natural selection occurs under conditions of globally weakened MCM reserves, and the concomitant accumulation of DNA damage from S-phase and M-phase malfunctioning, that leads to loss of growth control and eventual tumor formation.,24–26 In this sense, proper reserve MCM management is tumor-suppressive, while reserve MCM mismanagement is tumor-promoting (or tumor-driving).A clear demonstration of how mismanagement of reserve MCM complexes can produce a tumor-driver effect has been shown in an elegant set of studies using a transgenic mouse model in which MCM hexamers are destabilized due to a specific acquired mutation in the  The reduced MCM availability in cells, which would likely limit the reserve MCM complement, leads to replicative stress, changes in replication fork speed, DNA damage, and chromosome breaks that collectively contribute to the functional decline in HSC activity. Therefore, MCM reduction and consequent genomic instability can elicit biological effects separate from tumor promotion.A similar situation arises for MCM loss in aging hematopoietic stem cells (HSC), which functionally decline in activity over time. Such HSC display elevated levels of replicative stress as a result of diminished MCM expression.2.4.Mcm4 mutant mice are tumor prone, yet are not subjected to external stresses for DNA replication that stall forks, one might ask why there is evidence of stalled forks in such mouse cells. Even more relevant, one might also ask why human tumors do not seem to arise with underlying mutations in Mcm genes that weaken reserve MCM hexamers, display similar stalled forks, and produce genomic instability subjected to selection pressure at the cellular level that results in tumorigenesis. It is likely that normal DNA replication faces hurdles that create fork stalling situations, such as replication forks encountering heterochromatin, topological constraints of certain genomic regions, interference with transcriptional or related machinery, or impasses involving DNA lesions. These and other replicative stress conditions can be mitigated by cells using template switching, homologous recombination, fork reversal, or similar methods . Reee refs. ). Indeed3.Mcm mutations that adversely affect reserve MCM management, there is evidence that certain oncogene products do affect reserve MCM pools and CMG activation dynamics. The assembly of the CMG at pre-established MCM hexamer sites requires the function of the c-Myc protein (referred to hereafter as Myc), specifically in late-G1 when Cdc45 and GINS are recruited onto MCMs in mammalian cells. Myc interacts with many genomic sites, including promoter regions and intergenic regions, the latter of which are also regions where MCM hexamers are loaded onto DNA.,33 The genomic proximity of Myc to MCM complexes is associated with a function for Myc in recruiting the GCN5 and Tip60 histone acetyl-transferases (HATs), which cause chromatin decondensation necessary for Cdc45/GINS recruitment to MCMs  of the reserve pool of MCMs/CMGs. This concept is also nicely discussed in a review on Myc control of DNA replication.This molecular mechanism for Myc in regulating CMG assembly has important implications for tumorigenesis driven by Myc. It is estimated that ≈70% or more of human cancers have Myc overexpressed or deregulated in other ways that lead to elevated Myc activity in tumor cells.Mcm4Chaos3 mutant mice with weakened MCM-chromatin interactions, over-activation of reserve MCMs by Myc results in a similar functional outcome where the number of unused reserve MCMs are reduced due to Myc’s actions.–20 The reduction in unused reserve MCM capacity by Myc overexpression renders cells less effective at handling stalled replication forks.,19,21 Similar to that seen with the Mcm4Chaos3 mouse model, a Myc-driven deficiency in reserve MCM capacity concurrent with normal problems in Sphase that produce stalled replication forks will likely create an unstable genomic environment and DNA damage accumulation that leads to positively selected adaptations in mutated cells and tumorigenesis.,24 Since Myc is sometimes overexpressed in early stages of tumorigenesis,,40 it is possible that deregulation of Myc in benign tumors or early-stage cancers leads to genomic instability issues arising from reduced reserve MCM capacity, which facilitates selection and progression to later neoplastic stages. Indeed, Myc overexpression is known to be associated with acute onset of genomic instability.,41,42 producing replication stress, fork rate slowing, and fork asymmetry in a single cell cycle in a human cancer cell analysis. MCM/CMG over-use by Myc may be one underlying molecular explanation for these effects. It is important to note that the excessive activation of MCM/CMG helicases by Myc is not detrimental to ongoing DNA replication, and only produces shorter inter-origin distances due to more CMGs being active at one time.–20 It is only when conditions create stalled replication forks that an excess of Myc and consequent CMG over-activation will make for an aberrant situation. From this perspective, whereas Myc is the primary driver of tumorigenesis, Myc creates a second tumordriver by its actions: deficient pools of reserve MCMs that reduce the efficiency of recovery from stalled fork conditions, thereby promoting tumorigenesis due to increased DNA damage and genomic instability.This ability of overexpressed Myc, a tumor-driver itself, to overactivate and mismanage MCMs/CMGs has the propensity to create a new tumor-driver situation with regard to the reserve MCM capacity. Although conceptually different from the reduced reserve capacity of MCMs in the 4.Achilles’ Heel for a tumor, a Myc-driven weakness, and vulnerability. For example, in tumors with overexpressed Myc, the reserve MCM capacity may be reduced such that these tumors will display heightened sensitivity to chemical inhibitors that cause DNA replication fork stalling  in which recovery from fork-stalling drugs is impaired as a result of lowered reserve MCM/CMG capacity. We define this concept as the “reserve CMG helicase vulnerability.”Thus far, the over-activation of reserve MCMs/CMGs by Myc has been illustrated as a potential strength of the process that selects for malignant outgrowths, in that loss of reserve MCM capacity due to Myc’s actions produces an unstable genomic environment that drives tumor formation and tumor heterogeneity. However, the same MCM-debilitating process that can drive tumorigenesis is likely to also be an stalling . Indeed,A more intriguing concept based on this vulnerability in reserve MCM mismanagement due to Myc overexpression is that small chemical inhibitors against the CMG enzyme itself (CMGi) may prove useful in clinical approaches. If the MCM/CMG reserves are already debilitated due to Myc’s actions, then administering a CMGi has the propensity to make a bad situation worse, further inactivating the remaining (now limited) reserve CMG helicases and likely increasing the sensitization of tumor cells to fork stalling chemotherapy . Importa As discussed above, pancreatic ductal adenocarcinoma and colorectal cancer cells can be sensitized to clinically relevant chemotherapeutic  drugs when the reserve complement of MCMs is depleted using siRNA-mediated methods, mimicking the presence of a future CMGi as a proof-of-principle. Mechanistically, this sensitization derives from the fact that tumor cells with experimental MCM reserve loss have a reduced ability to recover DNA replication after replicative stresses induced by exposure to fork-stalling chemotherapy drugs. More importantly, under similar conditions of reserve MCM debilitation by siRNA, non-tumor cells are less sensitive to fork-stalling drugs than tumor cells. This indicates that tumor cells are selectively compromised for further loss of reserve MCM capacity during replicative stress, and that a therapeutic window likely does exist for CMGi intervention in cancer treatment. It is note worthy here that the genetic basis  for why the PDAC and CRC tumor lines tested in this study are vulnerable to CMG suppression is not known. Nonetheless, such studies and the potential for Myc to create a tumor-selective reserve MCM vulnerability in many human neoplasms strongly suggest that CMGi intervention may offer an innovative means of managing cancer in the clinic. We refer to this concept as the “reserve CMGi clinical hypothesis.”In support of these concepts, it has already been demonstrated that future CMGi are indeed likely to be effective against tumor cells in a selective manner, and sensitize tumor cells to chemotherapeutic drugs.5. In this case, the DNA repair/recovery ability of the tumor cells was already debilitated due to BRCA inactivation prior to exposure to a second repair-limiting drug, the PARPi. Given the potential or demonstrated weaknesses in the reserve MCM capacity of tumor cells that also limits repair/recovery, it becomes feasible to consider how a CMGi may be useful in similar future clinical regimens. For example, can a CMGi further debilitate a pre-existing weakness in MCM reserves and enhance chemotherapy for tumors carrying DNA repair deficiencies such as BRCA loss? Can a CMGi synergize with PARPi and DNA damaging drugs, or replace the PARPi drug in the clinical regimen? Do tumors with overexpressed Myc and debilitated MCM reserves provide a signature that predicts better responses to PARPi or other repair-limiting drugs in combination with fork-stalling chemotherapy? Can this be further enhanced with a CMGi included in patient treatment, concurrently or with different scheduling?A recent clinical trial found that pancreatic cancer patients who carry BRCA mutations are more sensitive to platinum therapy and PARP inhibitors (PARPi), indicating as a proof-of-principle that suppression of two DNA repair/recovery pathways is clinically advantageous for therapies that include DNA damaging drugs.Tp53 and RB1 deficient malignancies small cell lung cancer and osteosarcoma.,45 These and other possibilities argue for the need to develop drugs that can effectively and specifically inhibit the CMG helicase. However, it should be noted that, since loss of reserve MCM/CMG capacity has the potential to be a tumordriver, chronic CMGi exposure in the clinic might promote secondary malignant growths. As with other chemotherapy drugs that can similarly cause secondary neoplasms, this would need to be factored into the design of clinical regimens in treating patients.There are additional clinical considerations that justify anticancer intervention with a focus on targeting the CMG helicase. Recalcitrant metastatic lesions that fail to respond to conventional or personalized drug regimens due to biological changes from the primary lesions may yet retain MCM reserve vulnerabilities due to overexpressed Myc that sensitizes the distal lesions to CMGi. Likewise, tumors that fail first-line therapies may be suitable for second-line approaches involving CMGi due to genetic conditions such as Myc deregulation that affect MCM management. Examples of the latter include the For clinical utility, the CMG helicase needs to be amenable to drug targeting with small chemical moieties, and preferably in a unique manner that is not easily overcome through biological changes in tumor cells. One way to target the CMG is through an indirect approach, by inhibiting druggable enzymes that are required for CMG assembly or activation. For example, Cdc7 is a kinase required for CMG assembly, and several groups have developed Cdc7i that are in early clinical trials see . However,11,46,47  motif on Mcm7 important for ribose binding, phosphate interactions, and ATP hydrolysis.,11 Development of inhibitors against any of the ATPase clefts of the CMG will render the entire helicase inactive based on multiple studies of related CMG helicases in eukaryotes.,11,46,47 Such inhibitors will also be able to target not only functional CMG helicases, but also non-functional dormant MCM hexamers (reserves). Toward this end, there is a report of successful inhibition in vitro of the yeast MCM complex using fluoroquinolone based drugs. However, the dosages used were very high and the ability of these compounds to inhibit the purified hCMG enzyme has yet to be determined experimentally.Although the Cdc45 and GINS subunits are not enzymatically active in terms of targetable clefts, the hexameric MCM ring of the CMG has six targetable ATP-binding and hydrolyzing domains6,47 see . These AThe CMG helicase is the primary replicative helicase used for DNA replication within replisomes, and the CMG has no redundant functional counterpart. Therefore, it is difficult to envision a clinical situation where tumors can circumvent the CMG helicase (and a CMGi) by using an alternative enzyme or pathway. While it is possible that mutations could be selected for in tumors that limit the binding of CMGi to a particular cleft (as often happens with some kinases), this would only be achievable if the mutation does not render the helicase debilitated. Mutated and weakened CMGs are not likely to allow survival of tumor cells that already have MCM/CMG reserve deficiencies. In fact, the latter concept could be tested using a combination of catalytically-mutant MCM subunit expression simultaneously with siRNA-mediated targeting of a separate MCM subunit that achieves overall reduction in MCM hexamer numbers.6.,50 is upregulated by Myc, and Cyclin E deregulation has been shown to be associated with acute onset of genomic instability.,52,53 Interestingly, overexpression of Cyclin E causes a diminishing of the number of MCM hexamers that load onto chromatin/DNA prior to S-phase  Myc-driven over-activation of reserve MCMs, and iii) Cyclin E-driven reduction of MCM availability. Added to this are other effects of Myc and Cyclin E on creating irregular replication fork dynamics due to premature S-phase entry.Perhaps one explanation for why overexpressed Cyclin E has this effect on the reserve MCM pool derives from its ability to cause premature S-phase entry,e clinic  and 3. T suggesting the likelihood that other oncogenes may indeed also affect the dynamics of the reserve MCM pool, directly or indirectly. Or perhaps pathways not typically associated with oncogenic deregulation may be found to mismanage reserve MCM pools and thus act as drivers of carcinogenesis. Notably, tumors such as sarcomas often lack a specific oncogenic driver as an explanation for their development. Instead, some sarcomas  contain a large number of genomic rearrangements, chromosome losses, an euploidy, and hyper diploidy. Although a specific molecular reason for why these genomic issues arise is not known, one possibility is that the reserve MCM pool has been adversely affected at some stage in tumor development, rendering the genome unstable and causing selection for sarcoma outgrowth. Such a hypothesis might be welcome not only from a mechanistic point of view in terms of why such sarcomas develop, but also from a clinical translational perspective in which the mismanaged CMG reserves become a viable target in sarcomas for chemotherapeutic intervention due to predicted weaknesses in this process.Whether and how other oncogenes affect and/or mismanage reserve MCM pools remains to be determined. An elegant review of oncogene-induced replicative stress describes many cellular mechanisms that oncogenes influence leading to replicative stress as an outcome,7.The ability of Myc to over-stimulate conversion of MCMs into functional CMG helicases necessarily draws on the pool of reserve MCMs that could become active CMGs in times of need, such as during replicative stresses that occur normally, by oncogenes, or through insult with chemotherapy. This role for Myc in CMG (mis)management clearly provides at least one explanation for the loss of genome integrity that is common to many tumor types, and at the same time can explain how tumors can evolve under diminished reserve MCM capacity into heterogenous mixtures of cells at the genomic and physiologic levels. Interestingly, such a hypothesis for Myc would also suggest that this CMG deregulation would likely create a loss of viability for tumor cells as they evolve, due to stochastically acquired genomic issues that are not compatible with survival. Perhaps as a result of Myc-driven genomic instability/changes and selection pressure, some of these associated proteins will be found in future experiments to be deregulated in tumor cells, compensating for deficiencies in CMG function and further creating tumor heterogeneity in the surviving cells. However, even in the presence of such overriding events, the fundamental issue will remain that CMG fidelity has been weakened in tumors and remains an exploitable vulnerability due to the tumor’s need for a minimal CMG functionality to survive. Clearly, the future will uncover answers to these questions, which have important implications not only for our basic understanding of tumor evolution, but also for identifying other exploitable vulnerabilities.How might tumor cells evolve to override loss in CMG control by Myc and produce surviving cells? The CMG helicase is not a self-contained enzyme working in isolation, but rather is part of a large complex machine called a replisome, which incorporates proteins such as DNA polymerases, topoisomerases, cofactors, and other subunits.


Cancer is the second biggest cause of death in children in the US. With the development of chemotherapy, there has been a substantial increase in the overall survival rate in the last 30 years. However, the overall mortality rate in children with cancer remains 25%, and many survivors experience a decline in overall quality of life and long-term adverse effects caused by treatments. Although cancer cells share common characteristics, pediatric cancers are different from adult cancers in their prevalence, mutation load, and drug response. Therefore, there is an urgent unmet need to develop therapeutic approaches specifically designed for children with cancer. Nanotechnology can potentially overcome the deficiencies of conventional methods of administering chemotherapy and ultimately improve clinical outcomes. The nanoparticle-based drug delivery systems can decrease the toxicity of therapy, provide a sustained or controlled drug release, improve the pharmacokinetic properties of loading contents, and achieve a targeted drug delivery with achievable modifications. Furthermore, therapeutic approaches based on combining nanoformulated drugs with novel immunotherapeutic agents are emerging. In this review, we discussed the recently developed nanotechnology-based strategies for treating blood and solid pediatric cancers. In the United States, pediatric cancer is the second biggest cause of death for children under the age of 14, just exceeded by accidents . Since tCytotoxic chemotherapy utilizing DNA alkylating agents and antimetabolites, has been the most widely used cancer treatment in the past 50 years. Current chemotherapies produce a host of unintended effects that can pose lifelong implications for survivors, such as severe sequelae and decreased quality of life. Common side effects of current chemotherapies include fatigue, nausea, diarrhea, mouth sores, hair loss, and anemia. These apoptosis-inducing therapies cannot differentiate rapidly dividing normal cells from cancerous cells causing systemic toxicity. Furthermore, chemotherapy is typically administered daily through oral or intravenous injection, which constrains patients to daily medical appointments or inpatient care and further reduces their quality of life.A deeper understanding of cancer biology in recent decades has accelerated the design of molecules that target and inhibit proteins  and pathways  that are crucial for tumor growth or contribute to cancer cell proliferation . In the Cancer is an extremely heterogeneous disease with complex and tissue-specific impairments from genetic, epigenetic, and environmental factors. Understanding the critical differences between pediatric and adult cancers is integral for investigating the underlying pathophysiologic and molecular mechanisms for developing new diagnostic/therapeutic approaches .First, types of cancers are different between children and adults. Childhood cancers are not triggered by lifestyle as in adults, and very few are inherited from parents. Although the risk of developing most types of cancer significantly increases with age, there are several exceptions. For example, while brain and bone cancers are rare in adults, they have much higher rates in children ,10. SimiSecond, even though cancers in children tend to respond better to therapy, children and adults differ in genitourinary pH and transit, intestinal motility and conjugation, and transport of bile salts. All of these factors affect the metabolism of chemotherapeutic drugs. Pediatric tissues and organs are immature, and because children are rapidly developing, they have higher metabolic rates. Treatment dosages that are safe to administer to adults are often severely toxic to a child’s developing organs, which absorb, distribute, and eliminate substances more rapidly than the same type of organ in an adult. Finally, genetic stability in children and adults is also different, which affects mutation rates in cancer . In a stFor the above reasons, chemotherapy doses in children cannot be simplified by the direct adjustment to the body weight/surface, as they often are in adults. Therefore, there is an urgent unmet need to develop therapeutic methods specifically designed for children with cancer, which may provide clinicians with more powerful weapons to treat them.Significant differences between the participation of children and adults in clinical trials using nanocarriers are found through the search engine ClinicalTrial.gov. There are 1523 studies for liposome as treatments for cancer in adults, while there are just 364 studies for all age groups (including children). If we search for “pediatric” and “liposome”, 33 results were obtained, and only 11 of them are for pediatric cancer, with 10 studies being specially designed for children (excluding adults), as shown in While clinical trials in children are more complicated due to scientific, ethical, and technical factors that have stunted research progress in recent years, the lack of studies about drug disposition in pediatric patients poses a key difficulty for clinical applications. The extrapolation of adult dosing is often used to determine the dosage for pediatric patients. However, the pharmacokinetic and pharmacodynamic properties of roughly 70% of drugs prescribed to pediatric patients have not been appropriately studied in children, necessitating the development of new approaches for determining the dosage for pediatric patients . Over the past several decades, astounding progress has been made in the development of chemotherapy and the emergence of novel treatments for the adults, such as immunotherapy and combination therapy. Nevertheless, conventional anti-cancer treatments can have late side effects or cause severe long-term health problems that are not well studied once former pediatric cancer patients mature into adults . ConsequOne of the significant recent developments aimed to advance pediatric research into clinical development is the Research to Accelerate Cures and Equity for Children Act (RACE for Children Act) ,21,22 anNanotechnology is an emerging interdisciplinary strategy in cancer diagnosis and treatment. Nanotechnology has rapidly developed over the past ten years and has been intensively applied in fields of engineering, medicine, biology, and chemistry. For example, nanoparticles are widely used as a vehicle for the delivery of drugs as well as vaccines, or directly act as therapeutic agents for certain diseases ,27. MoreNanoparticles, defined as particles ranging from 1 to 1000 nm, have many unique properties compared to larger particles, and have been widely studied for their potential as anti-cancer drug delivery platforms , top. NaAlthough the anti-tumor efficacy of traditional chemotherapy drugs, such as anthracyclines or DNA alkylating agents, is well proven, the risk of toxicity in pediatric patients creates clinical limitations. Many promising drugs, such as the cyclin-dependent kinase 12 (CDK12) inhibitor dinaciclib, have limited clinical application because of their short half-life and high toxicity . In addiTumors can induce the growth of blood vessels to supply their cells with necessary nutrients and oxygen supply, resulting in highly disorganized and abnormal vascular networks containing poorly aligned, defective endothelial cells with wide fenestrations. These tissues lack a smooth muscle layer and adequate lymphatic drainage and have innervation with a broader lumen and impaired functional receptors. Such features improve permeability and increase the accumulation of some nanoparticles, such as liposomes (EPR effect) , bottom Cancer cells have different metabolic activity and active signaling pathways than normal cells, which leads to a significant divergence in surface receptor expression ,40,41. MA variety of types of nanoparticles developed for drug delivery or as diagnostic agents are described below.Metallic nanoparticles range from 1 to 100 nm in size and are frequently used as drug carriers and bioimaging agents. They are useful carriers based on their physicochemical properties, high stability, high reactivity, and photothermic and plasmonic properties . Gold naPolymeric micelles, dendrimers, and liposomes are widely used nanocarriers of hydrophobic drugs used to enhance aqueous solubility and prolong the half-life of chemotherapeutic agents in circulation. These nanoparticles are usually 10–100 nm in size and consist of a hydrophilic polyethylene glycol (PEG) outer shell and a hydrophilic core ,46. TheySuperparamagnetic iron oxide nanoparticles have been thoroughly studied as inorganic nanocarrier systems for drug delivery ,48,49. ICarbon nanotubes can be functionalized with bioactive peptides, proteins, nucleic acids, and drugs. When employed in this fashion, they are not immunogenic and display low toxicity in drug delivery. Carbon nanotubes have a high propensity for crossing cellular membranes, resulting in high levels of cellular uptake . NanotubQuantum dots range from 2 to 10 nm and are nanometric semiconductors with distinctive optical properties, including high quantum yield, size-tunable light emission, and good chemical and photo-stability. They are used for fluorescent imaging with increased transmission of visible light through biological tissue for diagnostic purposes, drug delivery, and optical agents in sensor systems of biomarkers. Some quantum dots that contain heavy metals such as cadmium and mercury exhibit high levels of toxicity; this can be reduced by functionalizing the surface of quantum dots with biocompatible molecules . More precise dosing in preclinical studies compared to free drugs;Higher dose with less toxicity;Improved pharmacokinetic properties of drugs;More selective, antibody-targeted drug delivery for cancers with specific surface protein expression.Thus, nanoformulations can potentially overcome the deficiencies of conventional methods of administering chemotherapy and improve clinical results. Accordingly, the key advantages of the nanoformulations are: Leukemias account for about 28% of all pediatric malignancies, and are the most common cancers in children . The disLipid-based nanoparticles have been tested to treat leukemia. One study reported the use of lipid-based solid nanoparticles to deliver mitoxantrone and a P-glycoprotein (P-gp) inhibitor β-element to overcome multidrug resistance . P-gp ovImmunotherapy is another strategy for cancer treatment, in which immune cells are activated or enhanced in their ability to detect and kill cancer cells. One group explored the use of ionizable lipid nanoparticles to deliver mRNA for chimeric antigen receptor (CAR) T cell therapy . CAR is Magnetic hyperthermia is also applied in anti-tumor therapy by targeting magnetic nanoparticles (MNPs) to the tumor site. In this paper, a kind of leukemia targeting MNPs was developed by immobilizing the epithelial cellular adhesion molecule (EpCAM) antibody on the surface of MNPs (EpCAM-MNPs). EpCAM-MNPs can target and remove leukemia cells circulating in the bloodstream and were reported to decrease viability of human monocytic leukemia (THP1) cells by over 40% .Lymphoma is a type of cancer that originates from the lymphatic system, enlarging the lymph nodes and metastasizing to other tissues via the lymphatic fluid. After leukemias and brain tumors, lymphoma is the third most common form of cancer among children. According to the National Cancer Institute, around 2200 people under the age of 20 are diagnosed with lymphoma in the United States every year. Hodgkin lymphoma and non-Hodgkin lymphoma comprise approximately 15% of all childhood malignancies . The antAnaplastic large cell lymphoma (ALCL), the most common T-cell pediatric lymphoma, has an active pathogenic ALK oncogene and shows a high level of cell surface expression of CD30. Zeng et al. reported a precision therapy for ALCL using nanoparticles made with RNA-based CD30-specific aptamers (Aptamer—synthetic oligonucleotide or a peptide chain which attaches to a specific target) and loaded with ALK oncogene-specific siRNA and doxorubicin. The conjugated aptamers allowed the nanoparticles to specifically target ALCL cells, while the loaded gene therapy agent siRNA and the chemotherapy agent doxorubicin enhanced their cancer-killing ability .The overactivation of the PI3K/mTOR signaling pathway in non-Hodgkin lymphoma made the corresponding inhibitor BEZ235 a very promising treatment. However, it was withdrawn from early phase clinical trials due to its off-target toxicity and poor solubility. To solve these problems, adding specific antibodies on nanoparticles as targeted drug delivery was a successful strategy. Kin Man Au et al. developed a nanoparticle conjugated with two antibodies, anti-CD20 and anti-Lym1, as tumor-targeting components and loaded them with the PI3K/mTOR inhibitor BEZ235 to treat non-Hodgkin lymphoma . Dual anIn addition to the delivery of small-molecule chemotherapy agents, using nanoparticles to deliver nucleic acid has been explored as an immediate treatment for cancer cells. A recent study shows the lipid nanoparticle-based delivery of siRNA as interference therapy to attack mantle cell lymphoma by silencing their mRNA associated with cancer cell proliferation . To overBone cancers occur mostly in older children and teens and account for about 3% of all pediatric cancers . OsteosaThe current standard treatment incorporates presurgical chemotherapy to shrink tumors, surgical resection, radiation therapy to remove what cannot be surgically resected, and post-surgery chemotherapy to lower the chance of relapse . HoweverAs with other cancer treatments, nanocarrier delivery to osteosarcoma tumors is an emerging research field aimed at increasing targeted drug delivery and decreasing the necessary dosage and cellular toxicity. This is achieved either through passive delivery, relying on the EPR effect, or active delivery, taking advantage of the osteosarcoma cancer environment’s acidic pH and nanoparticle surface modification.Liposomes have been the most broadly studied vehicle to target osteosarcoma due to their biocompatibility and surface modification ability . SpecifiRNAi therapies such as microRNAs and siRNAs have shown promise in downregulating proteins produced by osteosarcoma cells and could be effective in conjunction with chemotherapies ,72. HoweEwing sarcoma is another common malignant bone tumor that primarily affects adolescents and young adults. While it most frequently presents as a bone tumor, it can also develop in connective tissue and soft tissue surrounding bone. Surgery is the most common method for removing Ewing sarcoma, while chemo- and radiation therapy are usually performed to shrink the tumor before surgery or prevent metastasis and recurrence after surgery. Current protocols include five chemotherapeutic agents , four of which induce DNA damage, as does radiation therapy ,79,80,81Some research has suggested that using liposomes to deliver chemotherapy for Ewing sarcoma can decrease toxicity and increase drug circulation. In one report, nanoliposome formulas encapsulating the PARP1 inhibitor talazoparib increased the tolerated dose compared to oral administration ,85. AnotThe simultaneous delivery of two or more drugs that may further sensitize cancer cells  is also a strategy for cancer treatment and recurrence prevention. The Pediatric Preclinical Testing Program (PPTP) identified this synergistic activity by using talazoparib plus temozolomide to treat Ewing sarcoma . HoweverAnother study tested the use of a hydrolyzed galactomannan (hGM)-based amphiphilic nanoparticle for selective intratumoral accumulation in pediatric sarcoma . This seEWSR1-FLI1 at the mRNA and protein levels in a serum-containing medium. Diamond nanocrystals also display fluorescence properties that result from the creation of a nitrogen-vacancy color center inside the nanodiamond matrix. This property means they could be used for tracking throughout the lifespans of cells and organisms.Alhaddad et al. reported the use of a novel diamond nanoparticle coated with cationic polymers to deliver interfering RNA to Ewing sarcoma cells . In thisLipoproteins are also appropriate materials for nanoparticle synthesis. Bell et al. employed biomimetic high-density lipoprotein (HDL) nanoparticles. These bind to HDL receptors and scavenger receptor type B-1 (SCARB1), which in turn deprives cells of natural HDL and their cholesterol stores, and blocks cell proliferation. In medulloblastoma and hedgehog-driven Ewing sarcoma tissues, this strategy depleted the populations of cancer stem cells. Furthermore, HDL nanoparticles disrupted cell colony formation in medulloblastomas . This stBrain cancers comprise the second most common cancer in children, making up about 26% of all pediatric cancers . Brain cThe blood–brain barrier (BBB) is a dynamic interface that separates the brain from the circulatory system to protect the brain from potentially harmful chemicals and pathogens and regulate the transport of essential nutrition to maintain a stable microenvironment . The BBBOverwhelmingly, the literature suggests that molecular size plays a vital role in BBB penetration. However, molecular size does not necessarily interfere with BBB permeability . Some smNanotechnology has enabled significant progress in delivering therapeutic agents across the BBB. Materials such as gold, lipids, and proteins are being investigated as cytostatic agents or drug carriers to treat or diagnose brain cancer. Nanoparticle size and surface are two main factors that affect the ability to cross the BBB. Large particles (over 150 nm in diameter) tend to be blocked by it, whereas a slightly positive surface charge can be favorable for particle–endothelial cell binding . AntibodApart from diffusion, ligand-induced transcytosis is also being tested for delivering drugs across the BBB. While BBB is a highly selective semipermeable endothelial cell border, the unique protein transporters expressed on its membrane that allow necessary nutrients to maintain brain homeostasis provide ideal targets. The transferrin receptor (TfR) is responsible for transporting iron into the brain parenchyma to maintain appropriate iron levels needed for brain metabolism, neural conductivity, and overall brain function . The TfRSimilarly, cell-penetrating peptides (CPPs), which facilitate the cellular uptake of molecules ranging from nano-size particles to small chemical compounds to large fragments of DNA, have been used to penetrate the BBB. Wang et al. tested the efficacy of CPPs and transferrin modified liposomes (Tf-LPs) loaded with doxorubicin to treat glioma. This nanoparticle, which was 120 nm in size and had a zeta potential of 6.81 mV, showed enhanced cellular uptake and reduced toxicity in two types of glioma cells compared to free doxorubicin .Another promising marker for malignant glioma is the amplification of the epidermal growth factor receptor (EGFR) expression with a frequency of about 50% ,107. ErbImmunotherapy is more commonly used for brain tumors than for other tumor types because of the brain’s unique environment, which can be regarded as an immune-privileged site separate from the rest of the body that prohibits immune cells from entering ,112,113.Glioma is one of the most common brain cancers in children and adolescents, and originates from glial cells, which support and nourish nerves in the brain . A folacTfRs are overexpressed in both BBB endothelial cells and gliomas. Fan et al. reported a trans-BBB delivery strategy that used Human H-Ferritin and L-Ferritin protein-covered iron oxide nanoparticles (HFns) to target the TfRs of the BBB endothelial cells and induce transcytosis  Figure . The nanMedulloblastoma is a common pediatric brain tumor located in the cerebellum, the lower back part of the brain that controls coordination, movement, and balance. Once established, medulloblastoma tends to spread to other parts of the brain through the cerebrospinal fluid, but rarely spreads to other body tissues. It occurs at any age, but most likely to happen in childhood, and is rare in adults. Choi et al. explored the strategy of suicide gene therapy for pediatric brain cancer medulloblastoma. Poly(beta-amino ester) nanoparticles were developed to deliver plasmid DNA encoding the suicide gene of herpes simplex virus I thymidine kinase . The delKim et al. engineered high-density lipoprotein-mimetic nanoparticles with an enhanced stability and targeting ability for treating the sonic hedgehog (SHH) subtype of medulloblastoma . ApolipoRadiotherapy is an integral component of cancer treatment. However, the radiation-induced adverse sequelae and resistance create clinical limitations. Thus, combining radiotherapy with different therapeutic agents that block specific DNA repair pathways could achieve a better therapeutic efficacy than monotherapy with a lower radiation dosage that minimizes potential adverse effects. Kievit et al. reported a strategy to sensitize pediatric tumor cells, including medulloblastoma and ependymoma cells, to radiotherapy by the nanoparticle-based delivery of siRNA; this knocks down the expression of Ape1, an enzyme involved in the base excision repair pathway . This suRetinoblastoma makes up 3% of all childhood cancers, and in severe cases, it can cause blindness and metastasis beyond the eye if not treated promptly and effectively ,126. OcuResearchers explored the delivery of natural therapeutics through nanoparticles to treat retinoblastoma. One study utilized polymeric nano-micelles as a delivery method to improve the water solubility of celestrol, a Chinese herb that displays the inhibition of angiogenesis-mediated retinoblastoma growth in murine models . SimilarWilms tumor is the most common form of pediatric kidney cancer and accounts for 5% of all pediatric cancers . NanomedNanomedicine is still a relatively new field, and new oncology applications are being developed at exponential rates. The applications discussed above could be modified to treat several rare yet consequential pediatric cancers. Hepatoblastoma, hepatocellular carcinoma, pleuropulmonary blastoma, and tracheobronchial tumor together account for less than 2% of all pediatric cancers and require further exploration into nano-applications and diagnostics and treatment.With the maturity of novel anti-cancer therapy, more and more nanoparticle-based drugs are also being approved by the FDA, improving outcomes for adult cancer patients. Nonetheless, effective strategies for treatment-resistant pediatric cancers remain elusive and only a few drugs are now approved for pediatric patients. Currently, pediatric cancer is commonly treated with chemotherapy, which triggers potential severe side-effects and causes toxicity to normal tissues. With different metabolic rates and immature organs, the tolerated dose is also different in children and adults, making it more challenging to identify the optimal dosage. To prevent the drug resistance often brought about by monotherapy, the combination of two or more chemotherapy agents changed little over the past two decades. However, organs and tissues grow rapidly in children, which means that they can respond differently to the medication at different developmental stages. As a result, pediatric cancer survival rates remain low. What is worse, a large portion of survivors suffer from short- or long-term adverse effects, such as nephrotoxicity, cardiotoxicity, infertility, and deafness. Targeted therapy is studied extensively and believed to be a promising strategy to overcome off-target toxicity associated with such diseases. Nanoparticles can easily achieve this goal by conjugating the antibodies or specific peptides against the target proteins. This emerging field is of high interest to researchers in academia and pharmaceutical companies, leading to tremendous development progress. All of the nanoformulations discussed in this review are shown in


Wnt signaling and germ cell-specific factors involved in the regulation of germ cell regeneration.Germ cell acts as a link between transfer of genetic information and process of species evolution. Defects or malformations of germ cells can lead to infertility or tumors. Germ cell regeneration is one of the effective ways to treat the infertility. Therefore, it is of great scientific and clinical interests to dissect the cellular and molecular mechanisms underlying germ cell regeneration. Progress have already been achieved in germ cell regeneration using model organisms for decades. However, key open issues regarding the underpinning mechanisms still remain poorly understood. Zebrafish is well known for its powerful regenerative capacity to regenerate various tissues and organs. Recently, advances in genomics, genetics, microscopy, and single cell technologies have made zebrafish an attractive model to study germ cell development and regeneration. Here we review recent technologies for the study of germ cell regeneration in zebrafish, highlight the potential of germline stem cells (GSCs) in the contribution to reproductive system regeneration, and discuss the  Infertility is estimated to affect more than 186 million people in the world . In the Danio rerio) has remarkable regenerative capabilities and becomes one of the most widely used vertebrate models for regenerative studies. Zebrafish is capable of regenerating a range of tissues including fin  transgenic line zebrafish females at 28 day post-fertilization (dpf) for 2 weeks caused infertility due to complete apoptosis of their germ cells (Tg(zpc:g4vp16/uas:nfsb-mcherry) transgenic background females and found that a large number of oocytes were killed, but 1 month later the ovaries could recover completely to regain their reproductive functions (Tg(ziwi:CFP-NTR) transgenic background females reverted into the fertile males after most of ovarian gem cells were ablated by MTZ treatment (Tg(vasa:dendra2-NTR) transgenic line females failed to regenerate and finally reverted into the infertile males when the early stage germ cells were ablated by MTZ (Tg(asp:GPF-NTR), Tg(sam:GPF-NTR) and Tg(odf:GPF-NTR) transgenic background zebrafish  are consistently present in the animal testes and support spermatogenesis. However, whether the mature ovaries of all animals have GSCs or oogonial stem cells (OSCs) remains to be determined. In invertebrate ovaries, the existence of GSCs has been reported by many articles, and research . In verteriments . Recent eriments . For exa ovaries . Zebrafi of GSCs . Moreovet 75 dpf   and differentiate into oocytes through mitosis and meiosis, which is known as oogenesis. Many genes were reported to be involved in the regulation of oogenesis in zebrafish. ebrafish . ziwi anebrafish , 2008. Ment fish . ca15b iogenesis . Furtherand GSCs .zpc only labeled the oocytes, GSCs in the adult Tg(zpc:g4vp16/uas:nfsb-mcherry) transgenic background females were not ablated by MTZ. Therefore, their ovaries were able to recovered completely to regain their reproductive function due to the proliferation of the resident GSCs  transgenic females were not completely ablated by MTZ. Thus, these females reverted into the fertile males  by MTZ treatment. These females failed to regain their reproductive functions, indicating that zebrafish ovaries have no regeneration without GSCs  is expressed in germ cells and its’ mutation leads to failure of GSC establishment and fertility (Many factors were identified to be germ cell-specific genes and involved in germ cell specification and maintainace in zebrafish. These molecular factors are also primarily conserved and may contribute to germ cell regeneration. ntenance . Loss ofsurvival . piwil1  meiosis , 2008. aertility . Based oIn conclusion, progress have been achieved in germ cell regeneration by using zebrafish as an animal model and contributed to the development of this field. However, there are still many unanswered questions in the cellular and molecular mechanisms underlying germ cell regeneration. For example, there is little information on what other cells are involved in the regulation of germ cell regeneration except for GSCs. Furthermore, the interaction between somatic cells and germ cell regeneration, as well as the molecular mechanisms regulating self-renewal and differentiation of GSCs and reproductive regeneration, are still poorly understood. In addition, what factors of injury environment stimulate GSCs behaviors is worth to be explored during regeneration. To answer these questions, some of sequencing technologies such as single cell sequencing and epigenetic testing, which include DNA methylation, histone modification and micro-RNA, will need to be utilized for regeneration studies. Moreover, advances in live imaging technology for adult zebrafish will also help expand our toolbox for germ cell regeneration. With the development of these new technologies, important discoveries in the mechanisms of germ cell and reproductive regeneration using the zebrafish model will be realized. An in-depth understanding of the mechanisms underlying germ cell regeneration in species with highly regenerative abilities such as zebrafish may provide inspiration for therapeutic strategies in mammalians reproductive disease.ZC conceived and carried out the literature review research, designed the figures and diagrams, and wrote the manuscript. QY conceived and carried out the literature review research. LL conceived and carried out the literature review research and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The lateral hypothalamus (LH) is classically implicated in sleep-wake control. It is the main source of orexin/hypocretin and melanin-concentrating hormone (MCH) neuropeptides in the brain, which have been both implicated in arousal state switching. These neuropeptides are produced by non-overlapping LH neurons, which both project widely throughout the brain, where release of orexin and MCH activates specific postsynaptic G-protein-coupled receptors. Optogenetic manipulations of orexin and MCH neurons during sleep indicate that they promote awakening and REM sleep, respectively. However, recordings from orexin and MCH neurons in awake, moving animals suggest that they also act outside sleep/wake switching. Here, we review recent studies showing that both orexin and MCH neurons can rapidly  change their firing when awake animals experience external stimuli, or during self-paced exploration of objects and places. However, the sensory-behavioral correlates of orexin and MCH neural activation can be quite different. Orexin neurons are generally more dynamic, with about 2/3rds of them activated before and during self-initiated running, and most activated by sensory stimulation across sensory modalities. MCH neurons are activated in a more select manner, for example upon self-paced investigation of novel objects and by certain other novel stimuli. We discuss optogenetic and chemogenetic manipulations of orexin and MCH neurons, which combined with pharmacological blockade of orexin and MCH receptors, imply that these rapid LH dynamics shape fundamental cognitive and motor processes due to orexin and MCH neuropeptide actions in the awake brain. Finally, we contemplate whether the awake control of psychomotor brain functions by orexin and MCH are distinct from their “arousal” effects. Based on behavioral effects of anatomically targeted lesions, the lateral hypothalamus (LH) has been long recognized as a key brain center in the control of appetite and arousal [reviewed in This article presents recent experimental evidence and interpretations linking these “non-canonical” LH functions to specific neuropeptidergic neural subsets of the LH, specifically orexin and MCH neurons. The overall aim of this review is to place a small number of recent studies from the authors’ laboratory in a broader context; for more exhaustive reviews of the LH the readers are referred elsewhere e.g., . First, Orexin neurons are known to be essential for stable wakefulness, and their loss produces the sleep disorder narcolepsy across mammalian species . MCH neuin vivo and in brain slices, and are differentially modulated by some indicators of body energy status, such as glucose  of orexin neurons activate around initiation of running bouts . Optogeno firing , produceo firing . In turno firing .in vivo (These experiments supply causal evidence for a role of orexin neurons in rapid sensorimotor control in the awake brain . This auin vivo , until tin vivo , clarifiAnother recent study examined the role of the awake activity of orexin neurons by optogenetic silencing of this activity during self-paced spatial exploration in awake mice . Optosilin vivo suggest that downstream, such actions of orexin neurons may involve orexin projections to action-selection control regions such as the nucleus accumbens (Optogenetics-assisted circuit mapping and combinatorial chemogenetic and pharmacological experiments ccumbens ; and upsccumbens . The extccumbens . Furtherccumbens . Consideccumbens , orexin ccumbens .Based on pioneering electrophysiological recordings from a small number of MCH neurons in head-fixed rats , for somin vivo, revealed that MCH neurons are under inhibitory GABAergic control by local LH GAD65 neurons (The first study carried out video-controlled closed-loop optogenetic silencing of MCH cell activity waves associated with self-paced exploration of novel objects . Importa neurons . Optogen neurons .These findings suggest that mice fail to recognize objects unless their MCH cell activity “marks” prior encounters with the objects, and that a GAD65→MCH LH circuit shapes the size of the memory-formation-gating MCH cell activity. The object recognition memory investigated in this study is important for normal life of mammals . EarlierThe second study probing the function of MCH neural signals during wakefulness was motivated by evidence, from fiber photometry LH recordings, that MCH neurons are activated by fear-inducing aversive events, namely electrical foot-shocks . OptogenWhile many details of the two studies differ , their cHow the transient awake signals on MCH neurons achieve these effects on memory is not yet clear. A number of cellular mechanisms have been suggested for how a transient wave of neural activity can induce a lasting transformation in the potential for future memory-related synaptic alteration, e.g., the synaptic tagging hypothesis . At the The studies reviewed above, including some with direct confirmation of global brain state by EEG/EMG recordings  make it First, are the awake actions of orexin and MCH neurons mediated by the neuropeptides they release or by GABA/glutamate that they also co-release? This has been addressed, in some cases, by orexin/MCH receptor antagonists, where the data imply that some memory-modulating effect of MCH neurons and exploration-guiding effects of orexin neurons, indeed rely on these neuropeptides . In manySecond, are “sleep-active” and “wake-active” orexin/MCH neurons the same or different neural subpopulations? This question has been technically difficult to answer, because this requires recordings of neural ensembles across brain states. However, in the LH, this is now achievable using GRIN lenses combined with head-mounted miniscopes or 2-photon microscopy . For oreThe third question, which is invariably raised at conferences and in article peer-reviews related to this topic, and relates to the title of this review, is of a more conceptual nature. It can be summarized as follows: are actions of orexin and MCH during sleep and wake fundamentally the same, and related to impact of these cells on “arousal”? For example, when orexin cells are optogenetically stimulated, do mice run more not because orexin cells are directly involved in motor control, but because mice are more awake/aroused? When MCH cells are active during object investigation, do mice then remember the object better not because MCH cells directly control fundamental molecular underpinnings of memory gating, but because MCH cells make mice more aroused and so better able to “take in” the object?This question can be contemplated at two levels, by considering specific experimental observations, or by considering our definitions of “arousal.” To the best of our knowledge, there is little evidence that MCH neurons promote arousal, only that they promote sleep . “IncreaThe classification of neural effects as “arousal effects” requires a specific temporal or anatomical definition of arousal, which seems to be largely missing from the current literature. The speed of orexin cell excitation by external stimuli can be as short as 34 ms, and orexin cell electrical excitation can cause movements as rapidly as 300 ms . ConsistBoth authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer TS declared a past co-authorship with one of the authors DB to the handling Editor.


Nanobiocides also can be applied for the prevention and treatment of viral infections specifically against COVID-19.The effectiveness of silver nanomaterials (AgNMs), as antiviral agents, has been confirmed in humans against many different types of viruses. Nanobiocides-based AgNMs can be effectively applied to eliminate coronaviruses (CoVs), as the cause of various diseases in animals and humans, particularly the fatal human respiratory infections. Mostly, these NMs act effectively against CoVs, thanks to the NMs’ fundamental anti-viral structures like reactive oxygen species (ROS), and photo-dynamic and photo-thermal abilities. Particularly, the antiviral activity of AgNMs is clarified under three inhibitory mechanisms including viral entry limitation, attachment inhibition, and viral replication limitation. It is believed that nanobiocide with other possible materials such as TiO Virus infections are a major global health challenge, particularly because the emergence of resistant and unknown viral strains and the complications related to extended use reduce the usefulness of postoperative antiviral therapies against pathogenic viruses –3. BasedMany scientists and pharmaceutical companies have commenced discovering a well-organized and effective way to protect human lives from the two arising epidemic infectious diseases caused by coronaviruses, such as SARS and MERS in the last two decades. Small molecules, common antiviral drugs, antibody-based drugs are the most promising tools for the treatment of viral infections . In the Regarding the capability of all new science and technologies, nanotechnology offers powerful versatile tools for the investigation, detection, prevention, and treatment of viral infections . The dimAs alluded to above, many types of NMs have been applied against COVs, however, the comprehensive investigation and inspection of engineered NMs like nanobiocides still remained. Therefore, we aimed to focus on summary points of nanobiocides-based AgNMs effects on COVID-19 as a novel prevention approach.http://www.cdc.gov/coronavirus/). Coronavirus has four genera containing alpha, beta, gamma, and delta. Seven types of betacoronaviruses family are potent to infect human and three of them cause severe symptoms . Bats and rodents are the natural reservoirs of different betacoronaviruses subgenera [In recent years, the CoV outbreak has spread all over the world causing epidemic and pandemic outbreaks of acute respiratory and infectious diseases  virus, and some resistant strains, such as IIIB, Eli, Beni, 96USSN20, and SaquinavirRV . TypicalSuch antiviral nanomaterials can specifically inhibit viral infectivity irreversibly by blocking the interaction between SARS-CoV-2 spike protein with the angiotensin-converting enzyme-2 (ACE2) receptor . AgNPs cThe use of reliable, low-cost, and rapid virucidal agents with long-term efficiency for SARS-CoV-2 prevention is the foremost priority. Nanobiocides, including metal nanoparticles and engineered nanomaterials, are effectively stabilized by using ionic surfactants typically with a negative charge to present high antiviral activity and stability in water. The surfactant molecule is dispersed in a colloidal liquid to enhance the stability for globular micelle reaching in nano-size . ColloidA recent study by Jeremiah et al. found that , naked AThe antiviral effects of AgNPs are size and concentration-dependent which depend on the cell type and also the type of AgNPs. Smaller particles have a higher toxic potential due to the greater surface area of interaction with the bound protein. Most studies have observed the antiviral efficacy of AgNPs at concentrations ranging between 10 and 100 ppm [Other metal nanoparticles composed of gold (Au) titanium (Ti), and zinc (Zn) have already shown great results against different types of viruses mainly thanks to their various antimicrobial activities that could be used on inanimate and nonbiological surfaces to efficiently control the ongoing COVID-19 pandemic .In recent years, Silica nanoparticles have been exclusively applied to control viral transduction , deliverApplying functionalized silica nanoparticles revealed the attachment of cell receptors to the virus envelope which was inhibited by a strong virus-nanoparticle interaction owning to the similarity in their hydrophobic/hydrophilic characters . MoreoveLikewise, organic nanoparticles for delivering antivirals as nanoencapsulated drugs have been applied to improve drug bioavailability and targeted antiviral activity and also to promote efficient drug delivery that could be alternatives to the development of safer treatments for COVID-19 and other viral diseases. Nanoencapsulated drugs through activating/deactivating intracellular mechanisms may be more efficient to make irreversible destruction to viruses and interfere with the virus' reproduction cycle .Graphene also showed a good capacity for viral inhibition. Graphene-based material has been revealed that can hinder the entry and replication of enveloped RNA virus (coronavirus) and DNA virus (herpesvirus) in their target cells , 47. Stu2 and carbon nanomaterials exclusively nano-graphene materials can produce a more efficient disinfectant for long-term stability and low toxicity compared to common disinfectant , short-time exposure (about 1 min), high stability, and low toxicity make them superior over the common disinfectants, such as ethanol, sodium hypochlorite, and hydrogen peroxide. An overview of nanobiocides’ advantages was displayed in Fig. We hope that our investigation could have successfully addressed the role of nanobiocides as a unique antimicrobial agent which can aid scientists and researchers with their valuable insights to conquer the obstacles related to the SARS‐CoV‐2 virus control. We also hope that these engineered NMs including metal and metal oxide NPs give us a suitable direction to provide exclusive antiviral therapies and avoid forthcoming pandemics close to the current COVID‐19.